Tumor antigen protein and use thereof

ABSTRACT

An inducer of cytotoxic T cells comprising as an active ingredient a protein which comprises the same or substantially the same amino acid sequence as that shown in SEQ ID NO: 2, or a peptide derived from the protein above is provided.

This application is a Divisional of co-pending application Ser. No.11/529,000 filed on Mar. 24, 2005, and for which priority is claimedunder 35 U.S.C. §120. application Ser. No. 11/529,000 is the nationalphase of PCT International Application No. PCT/PCT/JP03/12037 filed onSep. 19, 2003 under 35 U.S.C. §371. This application also claimspriority of Application No. 2002-282345 filed in Japan on Sep. 27, 2002under 35 U.S.C. §119.

TECHNICAL FIELD

The present invention relates to a tumor antigen protein. Moreparticularly, the present invention relates to use of a tumor antigenprotein PBF and a gene encoding the same in the field of cancerimmunology.

BACKGROUND ART

It is known that the cell mediated immunity, particularly a cytotoxic Tcell (hereinafter, referred to as “CTL”) plays a significant role in invivo rejection of tumor cells or viral infection cells. CTLs recognize acomplex between a tumor antigen peptide and a major histocompatibilitycomplex class I antigen, i.e., MHC class I antigen, which is referred toas “HLA antigen” in the case of human, on the cell surface of tumorcells, and attack and kill the cells.

Tumor antigen peptides are generated through the intracellularprocessing of intracellularly synthesized proteins specific for tumors(tumor antigen proteins) and degradated by proteases. The resultanttumor antigen peptides form a complex with MHC class I antigens (HLAantigens) in endoplasmic reticulum and transported to the cell surfacewhere said complex is presented as an antigen. CTLs, when recognize thecomplex presented as an antigen, exhibit the anti-tumor effects throughcytotoxic action or production of lymphokines. As a consequence ofelucidation of a series of such actions, a therapy of tumor patients hasbecome available, wherein a tumor antigen protein or peptide isadministered as an immunotherapeutic agent, i.e., cancer vaccine,thereby enhancing tumor-specific CTLs in the patient.

Typical examples of tumor antigen proteins include those described inImmunity, 10: 281, 1999, Table 1. Specific examples include melanosomeantigens such as melanocyte tissue specific proteins, for instance,gp100 (J. Exp. Med., 179:1005, 1994), MART-1 (Proc. Natl. Acad. Sci.USA, 91:3515, 1994) and melanosome proteins such as tyrosinase (J. Exp.Med., 178:489, 1993). Examples of tumor antigen proteins other thanmelanoma include HER2/neu (J. Exp. Med., 181: 2109, 1995), CEA (J. Natl.Cancer. Inst., 87: 982, 1995), and PSA (J. Natl. Cancer. Inst., 89: 293,1997), etc. However, tumor antigen proteins broadly applicable tocancers (tumors) including sarcomas such as osteosarcoma have not beenreported yet.

Papillomavirus binding factor (PBF, GenBank data base Accession No.AF263928) has been identified as a factor recognizing the E2 bindingsite of papillomavirus (Virology 293,103-117, 2002). However, nothinghas been known about the relationships between the PBF and tumors.

DISCLOSURE OF INVENTION

A purpose of the present invention is to provide use of a tumor antigenprotein PBF and a gene encoding the same in the field of cancerimmunology.

The present inventors have established osteosarcoma cell line OS2000from a patient of osteosarcoma and then a CTL cell line TcOS2000cl-303having cytotoxic activity against the OS2000 cells.

The inventors then prepared 293-EBNA-B55 and 293-EBNA-A24 cells forassay by introducing into 293-EBNA cell lines the HLA-B5502 gene (one ofHLA-B55) and the HLA-A2402 gene (one of HLA-A24), respectively. The293-EBNA-B55 or 293-EBNA-A24 cells were transformed with a cDNA clonepool of cDNA library prepared from OS2000, and the resultanttransfectants were treated with TcOS2000cl-303. The amount of LDHresulting from the cytotoxic effect of TcOS2000cl-303 was measured todetermine whether or not TcOS2000cl-303 reacted. After repeating anenormous number of screenings, the present inventors have finally foundthat papillomavirus binding factor (PBF, GenBank Accession No. AF263928)is a novel tumor antigen protein having CTL inducing activity. Thenucleotide and amino acid sequences of PBF are shown in SEQ ID NO: 1 and2, respectively. The fact that PBF serves as a tumor antigen protein hasneither known nor expected so far and is quite a novel finding.

The present inventors then confirmed that PBF comprises a tumor antigenpeptide region(s) capable of binding to an HLA antigen. The inventorsalso found that the PBF is extensively and highly expressed in sarcomasand renal cancer.

The tumor antigen protein PBF, tumor antigen peptides derived therefrom,or genes encoding the same can be used in vivo or in vitro as an inducerof CTL or a cancer vaccine, wherein they exert therapeutic orameliorative effects on tumors such as oseteosarcomas, renal cancer, andthe like. PBF is also useful as a marker for tumors such as sarcomas,renal cancer, and the like.

The present invention has been established on the basis of the findingsabove.

Thus, the present invention encompasses the followings.

(1) An inducer of CTL comprising as an active ingredient a protein whichcomprises the same or substantially the same amino acid sequence as thatshown in SEQ ID NO: 2.(2) The inducer of CTL of (1), wherein the protein which comprises thesame or substantially the same amino acid sequence as that shown in SEQID NO: 2 is selected from (a) to (d) below:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

(b) a protein comprising an amino acid sequence wherein one or moreamino acids are deleted, substituted and/or added in the amino acidsequence shown in SEQ ID NO: 2, and being characterized by that a cellsexpressing the protein is recognized by CTLs;

(c) a protein comprising an amino acid sequence having at least 70%sequence identity with the amino acid sequence shown in SEQ ID NO: 2,and being characterized by that a cell expressing the protein isrecognized by CTLs; and

(d) a protein being encoded by a polynucleotide capable of hybridizingto a complementary strand of polynucleotide encoding the amino acidsequence shown in SEQ ID NO: 2 under stringent conditions, and beingcharacterized by that a cell expressing the protein is recognized byCTLs.

(3) A partial peptide of a protein comprising the same or substantiallythe same amino acid sequence as that shown in SEQ ID NO: 2, whichpeptide is recognized by CTLs when bound to an HLA antigen.(4) The peptide of (3), wherein the protein comprising the same orsubstantially the same amino acid sequence as that shown in SEQ ID NO: 2is selected from (a) to (d) below:

(a) a protein comprising the amino acid sequence of SEQ ID NO: 2;

(b) a protein comprising an amino acid sequence wherein one or moreamino acids are deleted, substituted and/or added in the amino acidsequence shown in SEQ ID NO: 2, and being characterized by that a cellexpressing the protein is recognized by CTLs;

(c) a protein comprising an amino acid sequence having at least 70%sequence identity with the amino acid sequence shown in SEQ ID NO: 2,and being characterized by that a cell expressing the protein isrecognized by CTLs; and

(d) a protein being encoded by a polynucleotide capable of hybridizingto a complementary strand of polynucleotide encoding the amino acidsequence shown in SEQ ID NO: 2 under stringent conditions, and beingcharacterized by that a cell expressing the protein is recognized byCTLs.

(5) The peptide of (3) or (4), wherein the HLA antigen is HLA-A24 orHLA-B55.(6) The peptide of (5), which comprises an amino acid sequence shown inany one of SEQ ID NO: 6-55.(7) The peptide of (5), which comprises an amino acid sequence wherein,in the sequence shown in any one of SEQ ID NO: 6-45, the amino acidresidue at position 2 is substituted by tyrosine, phenylalanine,methionine or tryptophan, and/or the C terminal amino acid byphenylalanine, leucine, isoleucine, tryptophan or methionine.(8) An epitopic peptide comprising a peptide of any one of (3) to (7).(9) An inducer of CTL comprising a peptide of any one of (3) to (8) asan active ingredient.(10) An inducer of CTL comprising a polynucleotide encoding a proteincomprising the same or substantially the same amino acid sequence asthat shown in SEQ ID NO: 2.(11) The inducer of CTL of (10), wherein the polynucleotide encoding aprotein comprising the same or substantially the same amino acidsequence as that shown in SEQ ID NO: 2 is selected from (a) to (g)below:

(a) a polynucleotide comprising the base sequence shown in SEQ ID NO: 1;

(b) a polynucleotide comprising the base sequence at positions 337-1878of that shown in SEQ ID NO: 1;

(c) a polynucleotide comprising a base sequence encoding the amino acidsequence of SEQ ID NO: 2;

(d) a polynucleotide comprising the base sequence shown in SEQ ID NO: 3;

(e) a polynucleotide capable of hybridizing to a complementary strand ofany one of polynucleotides (a) to (d) above under stringent conditions,and being characterized by that a cell expressing a protein encoded bythe polynucleotide is recognized by CTLs;

(f) a polynucleotide comprising a base sequence having at least 70%sequence identity with a polynucleotide set forth in any one of (a) to

(d) above, and being characterized by that a cell expressing a proteinencoded by the polynucleotide is recognized by CTLs; and

(g) a polynucleotide encoding a protein comprising an amino acidsequence, wherein one or more amino acids are deleted, substitutedand/or added in the amino acid sequence encoded by any one ofpolynucleotides (a) to (d) above, and being characterized by that a cellexpressing the protein encoded by the polynucleotide is recognized byCTLs.

(12) The inducer of CTL of (10) or (11), wherein the polynucleotidecomprises a base sequence selected from those shown in SEQ ID NO: 1,position 337-1878 of SEQ ID NO: 1 or SEQ ID NO: 3.(13) A nucleic acid comprising a polynucleotide encoding a peptide setforth in any one of (3) to (8).(14) An inducer of CTL comprising the nucleic acid of (13).(15) A method for producing an antigen-presenting cell comprising thestep of bringing a cell having antigen-presenting ability into contactwith any one of following (a) to (d) in vitro:

(a) a protein comprising the same or substantially the same amino acidsequence as that shown in SEQ ID NO: 2;

(b) a nucleic acid comprising a polynucleotide encoding the protein of(a);

(c) a peptide set forth in any one of (3) to (8); and

(d) a nucleic acid comprising a polynucleotide encoding the peptide of(c).

(16) An antigen-presenting cell obtainable according to the method of(15).(17) A method for inducing a CTL comprising the step of bringingperipheral lymphocyte cells into contact with any one of following (a)to (d) in vitro:

(a) a protein comprising the same or substantially the same amino acidsequence as that shown in SEQ ID NO: 2;

(b) a nucleic acid comprising a polynucleotide encoding the protein of(a);

(c) a peptide set forth in any one of (3) to (8); and

(d) a nucleic acid comprising a polynucleotide encoding the peptide of(c).

(18) The CTL inducible by the method of (17).(19) An antibody which specifically binds to the polypeptide set forthin any one of (3) to (7).(20) A tumor marker comprising a polynucleotide and/or a complementarypolynucleotide thereof, which polynucleotide comprises at least 15contiguous nucleotides in the base sequence of a polynucleotide encodinga protein comprising the same or substantially the same amino acidsequence as that shown in SEQ ID NO: 2.(21) The tumor marker of (20), which is a polynucleotide and/or acomplementary polynucleotide thereof, which polynucleotide comprises atleast 15 contiguous nucleotides in the base sequence of SEQ ID NO 1 orSEQ ID NO: 3.(22) A tumor marker comprising at least 8 contiguous amino acids in theamino acid sequence of a protein comprising the same or substantiallythe same amino acid sequence as that shown in SEQ ID NO: 2.(23) The tumor marker of (22), which comprises at least 8 contiguousamino acids in the amino acid sequence shown in SEQ ID NO: 2.(24) A tumor marker comprising an antibody to a protein comprising thesame or substantially the same amino acid sequence as that shown in SEQID NO:2, or the antibody set forth in (19).(25) The tumor marker of (24) comprising an antibody to the proteincomprising the amino acid sequence shown in SEQ ID NO:2.(26) An HLA tetramer comprising a peptide set forth in any one of (3) to(7) and an HLA antigen.(27) A tumor marker comprising the tetramer of (26).(28) The tumor marker set forth in any one of (20) to (25), and (27),wherein the tumor is sarcoma or renal cancer.(29) A diagnostic agent for tumor comprising a tumor marker set forth inany one of (20)-(25), (27) and (28).(30) The inducer of CTL set forth in (1), (2), (9), (10), (11), (12) or(14), which is used as a cancer vaccine.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a graph showing the reactivity of CTL (TcOS2000cl-303) toEBNA-B55 or 293-EBNA-A24 measured by LDH release assay, wherein the cellwas transfected with 1B9.1H4 cDNA and allowed to express the same. Inthe figure, the abscissa axis represents the absorbance at 490 nm.

FIG. 2 is a graph showing the reactivity of CTL (TcOS2000cl-303) toEBNA-B55 or 293-EBNA-A24 measured by ⁵¹Cr release assay, wherein therespective cells were transfected with 1B9.1H4 cDNA and allowed toexpress the same. (A) and (B) show the results obtained using293-EBNA-A24 and 293-EBNA-B55 cells, respectively. In the figure, theabscissa axis represents E/T ratio and the ordinate axis cytotoxicactivity.

FIG. 3 is a graph showing the reactivity of CTL (TcOS2000cl-303) to293-EBNA-A24 measured by LDH release assay, wherein the cell wastransfected with 1B9.1H4 or PBF gene and allowed to express the same. Inthe figure, the abscissa axis represents the absorbance at 490 nm.

FIG. 4 is a graph showing the reactivity of CTL (TcOS2000cl-303) to293-EBNA-B55 measured by LDH release assay, wherein the cell wastransfected with 1B9.1H4 or PBF gene and allowed to express the same. Inthe figure, the abscissa axis represents the absorbance at 490 nm.

FIG. 5 is a graph showing the reactivity of CTL (TcOS2000cl-303) to293-EBNA-B55 measured by LDH release assay, wherein the cell was pulsedwith the peptide shown in SEQ ID NO: 46. In the figure, the abscissaaxis represents the absorbance at 490 nm.

FIG. 6 is a photograph showing the expression of a gene encoding tumorantigen protein PBF in various types of cells analyzed by the reversetranscription-PCR (RT-PCR). In the figure, OS2000 is an osteosarcomacell line, PBL is normal peripheral blood lymphocyte cell, EB-B is EBVtransform B cell, K562 is chronic myelocytic leukemia cell line, and293EBNA is human renal cell line transformed with adenovirus. SaOS, HOS,KIKU, Huo9 and NY represent osteosarcoma cell lines. HS-SYII, SW982 andFuji represent synovial sarcoma cell lines. HT1080 representsfibrosarcoma cell line. HS729T, A204 and RD represent rhabdomyosarcomacell lines. Further, A673, W-ES, NCR-EW2, SCCH196, SK-ES1 and RD-ES1represent Ewing's sarcoma cell lines. The upper panel represents theexpression of PBF gene, and the lower panel the expression of G3PDH genethat is used as a positive control.

BEST MODE FOR CARRYING OUT THE INVENTION 1) Protein of the PresentInvention

The protein contained in the inducer of CTL of the present invention,which protein may be referred to as “protein of the present invention”,comprises the same or substantially the same amino acid sequence as thatshown in SEQ ID NO: 2. The protein of the present invention may be aprotein originated from natural source (e.g., osteosarcoma cell line) ora recombinant protein.

The amino acid sequence shown in SEQ ID NO: 2 is registered with theGenBank database under Accession No. AF263928, and represents humanpapillomavirus binding factor (PBF) disclosed in Virology 293, 103-117(2002).

The aforementioned “a protein comprising the same amino acid sequence asthat shown in SEQ ID NO:2 (i.e., a protein comprising the amino acidsequence shown in SEQ ID NO:2)” specifically include a proteinconsisting of the amino acid sequence shown in SEQ ID NO:2 and a proteinconsisting of an amino acid sequence wherein the amino acid sequence ofSEQ ID NO:2 has an additional amino acid sequence attached to the Nand/or C terminus.

The aforementioned “a protein comprising substantially the same aminoacid sequence as that shown in SEQ ID NO:2” specifically include thefollowing proteins (a) to (c).

(a) a protein comprising an amino acid sequence wherein one or moreamino acids are deleted, substituted and/or added in the amino acidsequence shown in SEQ ID NO: 2, and being characterized by that a cellexpressing the protein is recognized by CTLs;

(b) a protein comprising an amino acid sequence having at least 70%sequence identity with the amino acid sequence shown in SEQ ID NO: 2,and being characterized by that a cell expressing the protein isrecognized by CTLs; and

(c) a protein being encoded by a polynucleotide capable of hybridizingto a complementary strand of polynucleotide encoding the amino acidsequence shown in SEQ ID NO: 2 under stringent conditions, and beingcharacterized by that a cell expressing the protein is recognized byCTLs.

Preferred examples include a protein consisting of a substantially thesame amino acid sequence as that shown in SEQ ID NO: 2. Examples of sucha protein consisting of a substantially the same amino acid sequence asthat shown in SEQ ID NO: 2 include the proteins (a′) to (c′) below.

(a′) a protein consisting of an amino acid sequence wherein one or moreamino acids are deleted, substituted and/or added in the amino acidsequence shown in SEQ ID NO: 2, and being characterized by that a cellexpressing the protein is recognized by CTLs;

(b′) a protein consisting of an amino acid sequence having at least 70%sequence identity with the amino acid sequence shown in SEQ ID NO: 2,and being characterized by that a cell expressing the protein isrecognized by CTLs; and

(c′) a protein being encoded by a polynucleotide capable of hybridizingto a complementary strand of polynucleotide encoding the amino acidsequence shown in SEQ ID NO: 2 under stringent conditions, and beingcharacterized by that a cell expressing the protein is recognized byCTLs.

The phrase “protein comprising an amino acid sequence wherein one ormore amino acids are deleted, substituted and/or added in the amino acidsequence shown in SEQ ID NO: 2” refers to so-called modified (variant)proteins produced artificially or allele variants present in a livingbody.

In this respect, there is no limitations regarding the number orposition of modification (mutation) in the protein as far as theactivity of the protein of the present invention is maintained. Criteriabased on which one can determine the number or position of amino acidresidue to be deleted, substituted and/or added without reducing theactivity can be obtained using a computer program well known in the art,such as DNA Star software. For example, the number of mutation wouldtypically be within 10%, preferably 5% of the total amino acid residues.Furthermore, the amino acid used for substitution preferably has similarcharacteristics to the one to be substituted in view of retention ofstructure, which characteristics include polarity, charge, solubility,hydrophobicity, hydrophilicity, amphipathicity, etc. For instance, Ala,Val, Leu, Ile, Pro, Met, Phe and Trp are classified into nonpolar aminoacids; Gly, Ser, Thr, Cys, Tyr, Asn and Gln into non-charged aminoacids; Asp and Glu into acidic amino acids; and Lys, Arg and H is intobasic amino acids. One of ordinary skill in the art can select anappropriate amino acid(s) falling within the same group on the basis ofthese criteria.

Examples of “a protein comprising an amino acid sequence having at least70% sequence identity with the amino acid sequence shown in SEQ ID NO:2” in (b) above include proteins comprising amino acid sequences havingat least about 70%, preferably, about 80%, more preferably, about 90%,and further more preferably about 95% sequence identity with the aminoacid sequence shown in SEQ ID NO: 2, and specifically, proteinsconsisting of a partial amino acid sequence of SEQ ID NO:2.

The term “sequence identity” herein used refers to the identity andhomology between two proteins. The “sequence identity” is determined bycomparing two sequences aligned optimally over the regions correspondingto the sequences to be compared. In this context, the both proteins tobe compared may have addition or deletion (e.g., “gap”) in theirsequences for optimum alignment. Such sequence identity can becalculated by preparing alignment using, for example, Vector NTI,ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)). Thesequence identity can be determined using software for sequenceanalysis, specifically, Vector NTI or GENETYX-MAC, or a sequencing toolprovided by a public database. The term “sequence identity” herein usedrefers to the identity and homology between two proteins. The “sequenceidentity” is determined by comparing two sequences aligned optimallyover the regions corresponding to the sequences to be compared. In thiscontext, the both proteins to be compared may have addition or deletion(e.g., “gap”) in their sequences for optimum alignment. Such sequenceidentity can be calculated by preparing alignment using, for example,Vector NTI, ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680(1994)). The sequence identity can be determined using software forsequence analysis, specifically, Vector NTI or GENETYX-MAC, or asequencing tool provided by a public database.

Examples of “a polynucleotide capable of hybridizing to a complementarystrand of polynucleotide encoding the amino acid sequence shown in SEQID NO: 2 under stringent conditions” in (c) include polynucleotidescomprising base sequences having at least about 40%, preferably, about60%, more preferably, about 70%, still more preferably about 80%,further more preferably about 90%, and most preferably, about 95%sequence identity with a polynucleotide encoding the amino acid sequenceshown in SEQ ID NO: 2. Specifically, examples include polynucleotidescomprising base sequences having at least about 40%, preferably, about60%, more preferably, about 70%, still more preferably about 80%,further more preferably about 90%, and most preferably, about 95%sequence identity with the base sequence of SEQ ID NO: 1, the basesequence at positions 337-1878 of SEQ ID NO: 1, or the base sequence ofSEQ ID NO:3. More specifically, examples include nucleic acidsconsisting of partial sequences of the base sequences of SEQ ID NO: 1,positions 337-1878 of SEQ ID NO: 1 or SEQ ID NO:3.

Hybridization can be conducted according to a method known per se or amethod equivalent thereto, for example, that described in a fundamentaltext such as “Molecular Cloning 2nd Edt. Cold Spring Harbor LaboratoryPress (1989)”, and the like. Also, it can be performed using acommercially available library according to the instructions attachedthereto.

The “stringent conditions” herein used can be determined on the basis ofthe melting temperature (Tm) of nucleic acids forming a complex with orbinding to probe as described in literatures (Berger and Kimmel, 1987,“Guide to Molecular Cloning Techniques Methods in Enzymology”, Vol. 152,Academic Press, San Diego Calif.; or “Molecular Cloning” 2nd Edt. ColdSpring Harbor Laboratory Press (1989), ibid.).

For example, hybridization can be carried out in a solution containing6×SSC (20×SSC means 333 mM sodium citrate, 333 mM NaCl), 0.5% SDS and50% formamide at 42° C., or in a solution containing 6×SSC (without 50%formamide) at 65° C.

Washing after hybridization can be conducted under a condition around“1×SSC, 0.1% SDS, 37° C.”. The complementary strand preferably remainsbound to the target sense when washed under such washing conditions.More stringent hybridization conditions may involve washing under theconditions of around “0.5×SSC, 0.1% SDS, 42° C.” and still morestringent hybridization conditions involve washing conditions of around“0.1×SSC, 0.1% SDS, 65° C.”, although it is not limited thereto.

The protein of the present invention has an activity of substantiallythe same quality as that having amino acid sequence of SEQ ID NO:2. Theterm “activity of substantially the same quality” refers to thecharacteristic future that cells expressing the protein are recognizedby CTLs, that is, the said cells exhibit reactivity to CTLs, in otherwords, the protein of the present invention or tumor antigen peptidederived therefrom activates or induces CTLs.

In this respect, the term “cells” are preferably those expressing HLAantigen. Accordingly, the said phrase “activity of substantially thesame quality” more specifically refers to the characteristics that, whenthe protein of the present invention is expressed in cells expressingHLA antigens such as HLA-A24 or HLA-B55, a complex between a tumorantigen peptide originated from the protein of the present invention andan HLA antigen is presented on the cell surface and consequently thecells are recognized by CTLs, in other words, CTLs are activated(induced).

Such characteristics of the protein of the present invention can beeasily determined by a method known or a method equivalent thereto, suchas ⁵¹Cr release assay (J. Immunol., 159: 4753, 1997), LDH release assayusing LDH Cytotoxicity Detection Kit (Takara Bio, Inc.), measurement ofcytokines, and the like. The detailed protocol of assay will hereinafterbe illustrated.

First, a host cell such as 293-EBNA cell (Invitrogen) is co-transfectedwith an expression vector comprising a DNA encoding the protein of thepresent invention and an expression vector comprising a DNA encoding HLAantigen. The DNA encoding HLA antigen includes a DNA encoding HLA-A24antigen or HLA-B55 antigen. Examples of DNA encoding HLA-A24 antigeninclude HLA-A2402 cDNA (Cancer Res., 55: 4248-4252 (1995), GenbankAccession No. M64740. Examples of DNA encoding HLA-B55 antigen includeHLA-B5502cDNA (GenBank Acc. No. M77777, J. Immunol., 148 (4), 1155-1162(1992)).

The transfection above can be conducted by Lipofectin method usinglipofectamine reagent (GIBCO BRL), and the like. Then, CTLs restrictedto the HLA antigen used are added and allowed to react, followed bymeasurement of various cytokines (for example, IFN-γ) produced by CTLsreacted or activated by, for example, ELISA. CTLs usable herein includethose prepared by stimulating peripheral blood lymphocytes with theprotein of the present invention (SEQ ID NO: 2) or those establishedaccording to the method of Int. J. Cancer, 39, 390-396, 1987, N. Eng. J.Med, 333, 1038-1044, 1995, or the like.

The CTL induction activity of the protein of the present invention canalso be examined in vivo by an assay where human model animals are used(WO 02/47474; Int. J. Cancer: 100, 565-570 (2002)).

The protein of the present invention can be prepared by a method knownper se that used for purifying protein from natural products (e.g.,osteosarcoma cell line, renal cancer cell line) or by a methodhereinafter described comprising culturing transformants carrying anucleic acid comprising a polynucleotide encoding the protein of thepresent invention.

2) Peptide of the Present Invention

The peptide contained in the inducer of CTL of the present invention,which peptide may be referred to as “peptide of the present invention”,is a partial peptide of the protein of the present invention as definedabove and, when bound to HLA antigen, is recognized by CTLs. Thus, thepeptide of the present invention may be any one existing at anypositions of the amino acid sequence of the protein of the presentinvention and of any length, as long as said peptide consists of apartial amino acid sequence of the protein of the present invention asdefined above and can form a complex with an HLA antigen that isrecognized by CTLs.

The peptide of the present invention can be identified by synthesizing acandidate peptide, which is a partial fragment of the protein of thepresent invention, and subjected to an assay to examine whether or notCTLs recognize a complex between said candidate peptide and HLA antigen,that is, whether or not the candidate peptide has the activity as atumor antigen peptide.

Synthesis of a peptide can be conducted according to processes generallyused in the field of peptide chemistry. Such a method can be found inliteratures including Peptide Synthesis, Interscience, New York, 1966;The Proteins, Vol. 2, Academic Press Inc., New York, 1976; PeptideSynthesis, Maruzen, Inc., 1975; Peptide-Gosei no Kiso to Jikken,Maruzen, Inc., 1985; and Iyakuhin no Kaihatsu (Zoku), Vol. 14, PeptideSynthesis, Hirokawa-syoten, 1991.

The method for identification of the tumor antigen peptide of thepresent invention will hereinafter be described in detail.

The regularity (motif) in the amino acid sequence of a tumor antigenpeptide that binds to HLA molecule and is presented has been elucidatedin relation to HLA-A1, -A0201, -A0204, -A0205, -A0206, -A0207, -A11,-A24, -A31, -A6801, -B7, -B8, -B2705, -B37, -Cw0401, -Cw0602. See,Immunogenetics, 41: p. 178, 1995, etc. For example, motifs for HLA-A24are known to have an amino acid sequence of 8-11 amino acids, whereinthe position 2 amino acid is tyrosine, phenylalanine, methionine ortryptophan, and the C-terminal amino acid phenylalanine, leucine,isoleucine, tryptophan or methionine (J. Immunol., 152, p 3913, 1994,Immunogenetics, 41: p 178, 1995. J. Immunol., 155:p 4307, 1994). As formotifs for HLA-A2, those listed in Table 1 are known (Immunogenetics,41, p 178, 1995, J. Immunol., 155: p 4749, 1995).

TABLE 1 2nd amino acid from amino acid HLA-A2 type N-terminus atC-terminus HLA-A0201 L, M V, L HLA-A0204 L L HLA-A0205 V, L, I, M LHLA-A0206 V, Q V, L HLA-A0207 L L *: All the peptides are 8-11 in aminoacid length.

Recently, it has become possible to search peptide sequences expected tobe capable of binding to HLA antigens via the internet using BIMASsoftware.

As for the length of the peptides, analysis of antigen peptides bindingto various HLA molecules revealed that it is generally about 8 to 14amino acids (Immunogenetics, 41: 178, 1995). However, in the cases ofHLA-DR, -DP, -DQ, peptides consist of 14 amino acids or more are known.

It is easy to select the peptide portions related to the motifs in theprotein of the present invention. For example, search for the sequencesexpected to be capable of binding to HLA antigen may be facilitated bymeans of BIMAS software. The peptide of the present invention can beidentified by synthesizing the selected candidate peptide by theabove-mentioned method, and examining whether or not said candidatepeptide binds to HLA antigen and is recognized by CTLs, that is, whetheror not the candidate peptide has an activity as a tumor antigen peptide.

Specifically, identification can be done by the method descried in J.Immunol., 154, p 2257, 1995. Thus, a candidate peptide is added tostimulate in vitro peripheral blood lymphocytes isolated from a humanpositive for an HLA antigen that is expected to present the candidatepeptide. When CTLs specifically recognizing the HLA-positive cellspulsed with the candidate peptide are induced, said candidate peptide ispossibly a tumor antigen peptide. The presence or absence of inductionof CTLs may be examined by, for example, measuring the amount of variouscytokines (e.g., IFN-γ) produced by CTLs in response to theantigen-presenting cells using ELISA or the like. Alternatively, theinduction of CTLs can also be examined by ⁵¹Cr release assay whereincytotoxicity of CTLs on antigen-presenting cells labeled with ⁵¹Cr ismeasured (Int. J. Cancer, 58: p 317, 1994).

Furthermore, the induction of CTLs can be examined by pulsing a cellsuch as 293-EBNA cell (Invitrogen) with a candidate peptide, to whichcell an expression plasmid expressing a cDNA encoding HLA antigen of atype expected to present the candidate peptide has been introduced,reacting the cell with CTLs restricted to HLA antigen of theaforementioned type that is expected to present the said candidatepeptide, and measuring various cytokines (e.g., IFN-γ) produced by theCTLs (J. Exp. Med., 187: 277, 1998).

Examples of HLA antigen include a HLA-A24 antigen and HLA-B55 antigen.To select HLA-A24-restricted tumor antigen peptide, HLA-A2402 cDNA(Cancer Res., 55: 4248-4252 (1995), Genbank Accession No. M64740) can beused as the cDNA encoding HLA antigen. To select HLA-B55-restrictedtumor antigen peptide, HLA-B5502 cDNA (GenBank Acc. No. M77777, J.Immunol., 148 (4), 1155-1162 (1992)) can be used as the cDNA encodingHLA antigen.

As for CTLs, in addition to those obtained by stimulating humanperipheral blood lymphocytes with a peptide, those established by amethod described in literatures (Int. J. Cancer, 39, 390-396, 1987; N.Eng. J. Med, 333, 1038-1044, 1995) may be used.

The in vivo activity of the peptide of the present invention can bedetermined by an assay which uses an animal model for human (WO02/47474, Int J. Cancer: 100, 565-570 (2002)).

In the above case, the regularity (motif) of the sequence of tumorantigen peptide is known; however, when the motif of a peptide isunknown, as in the case of HLA-B55, the tumor antigen peptide of thepresent invention can be identified according to the method describedin, for example, WO97/46676 or Example 3 below, only if CTL cell linescapable of recognizing a complex between said HLA-B55 and tumor antigenpeptide is available.

Specific examples of a peptide of the present invention include partialpeptides derived from the protein of the present invention consisting ofthe amino acid sequence shown in SEQ ID NO: 2, and capable of binding toHLA antigen and being recognized by CTLs. Preferred examples includepeptides capable of binding to HLA-A24 or HLA-B55 antigen, consideringthe HLA antigen to which the peptide of the present invention binds.

More specifically, examples of HLA-A24-binding tumor antigen peptideinclude a peptide (i.e., a peptide consisting of any one of amino acidsequences shown in SEQ ID NO: 6-45) having any one of amino acidsequences listed in Table 2 (9 amino acids) and Table 3 (10 aminoacids), and being recognized by CTLs when bound to HLA-A24 antigen.

TABLE 2 Position Amino acid sequence SEQ ID No. 145-153 Ala Tyr Arg ProVal Ser Arg SEQ ID NO: 6 Asn Ile 320-328 Asp Phe Tyr Tyr Thr Glu Val SEQID NO: 7 Gln Leu 254-262 Gly Phe Glu Thr Asp Pro Asp SEQ ID NO: 8 ProPhe 240-248 Lys Tyr Leu Gly Asp Ala Phe SEQ ID NO: 9 Gly Ser 12-20 ArgSer Leu Leu Gly Ala Arg SEQ ID NO: 10 Val Leu 30-38 Ala Ala Pro Pro SerGlu Pro SEQ ID NO: 11 Leu Leu 424-432 Ile Tyr Thr Ser Val Ser Trp SEQ IDNO: 12 Ala Ala 105-113 Thr Val Trp Leu Leu Glu Gln SEQ ID NO: 13 Lys Leu234-242 His Pro Gln Ala Ser Pro Lys SEQ ID NO: 14 Tyr Leu 440-448 LeuSer Pro Val Arg Ser Arg SEQ ID NO: 15 Ser Leu 279-287 Met Tyr Lys CysLeu Trp Pro SEQ ID NO: 16 Asn Cys 283-291 Leu Trp Pro Asn Cys Gly LysSEQ ID NO: 17 Val Leu 54-62 Cys Gln Glu Gln Pro Lys Glu SEQ ID NO: 18Val Leu 432-440 Ala Ala Pro Ser Ala Ala Cys SEQ ID NO: 19 Ser Leu101-109 Glu Gly Gln Val Thr Val Trp SEQ ID NO: 20 Leu Leu 169-177 MetAla Ala Met Val Leu Thr SEQ ID NO: 21 Ser Leu 129-137 Gly Pro Cys ProGln Ala Pro SEQ ID NO: 22 Pro Leu 354-362 Ala Pro Thr Pro Ser Met ThrSEQ ID NO: 23 Gly Leu 503-511 Arg Trp Lys Lys Ala Cys Gln SEQ ID NO: 24Arg Phe 29-37 Ser Ala Ala Pro Pro Ser Glu SEQ ID NO: 25 Pro Leu

TABLE 3 Position Amino acid sequence SEQ ID No. 84-93 Trp Tyr Gly GlyGln Glu Cys SEQ ID NO: 26 Thr Gly Leu 409-418 Ala Tyr Gln Ala Leu ProSer SEQ ID NO: 27 Phe Gln Ile 254-263 Gly Phe Glu Thr Asp Pro Asp SEQ IDNO: 28 Pro Phe Leu 118-127 Arg Val Glu Glu Val Trp Leu SEQ ID NO: 29 AlaGlu Leu 415-424 Ser Phe Gln Ile Pro Val Ser SEQ ID NO: 30 Pro His Ile81-90 Val Tyr Val Trp Tyr Gly Gly SEQ ID NO: 31 Gln Glu Cys 104-113 ValThr Val Trp Leu Leu Glu SEQ ID NO: 32 Gln Lys Leu 99-108 Trp Met Glu GlyGln Val Thr SEQ ID NO: 33 Val Trp Leu 503-512 Arg Trp Lys Lys Ala CysGln SEQ ID NO: 34 Arg Phe Leu 362-371 Leu Pro Leu Ser Ala Leu Pro SEQ IDNO: 35 Pro Pro Leu 295-304 Val Gly Ile Lys Arg His Val SEQ ID NO: 36 LysAla Leu 274-283 Asn Ser Val Lys Val Met Tyr SEQ ID NO: 37 Lys Cys Leu128-137 Gln Gly Pro Cys Pro Gln Ala SEQ ID NO: 38 Pro Pro Leu 404-413Ile Gln Ala Asp His Ala Tyr SEQ ID NO: 39 Gln Ala Leu 168-177 Met MetAla Ala Met Val Leu SEQ ID NO: 40 Thr Ser Leu 424-433 Ile Tyr Thr SerVal Ser Trp SEQ ID NO: 41 Ala Ala Ala 489-498 Val Tyr Gly Ile Glu HisArg SEQ ID NO: 42 Asp Gln Trp 439-448 Ser Leu Ser Pro Val Arg Ser SEQ IDNO: 43 Arg Ser Leu 431-440 Ala Ala Ala Pro Ser Ala Ala SEQ ID NO: 44 CysSer Leu 455-464 Gln Pro Ala Pro Ala Met Lys SEQ ID NO: 45 Ser His Leu

Further, HLA-B55-restricted tumor antigen peptide includes a peptidehaving the amino acid sequence: Cys Thr Ala Cys Arg Trp Lys Lys Ala CysGln Arg (SEQ ID NO: 46). In addition, a peptide consisting of 9-, 10- or11-amino acid portion of the peptide above, that is, a peptide havingany one of amino acid sequences listed below, which peptide can berecognized by CTLs when bound to HLA-B55 antigen, is also included.

(SEQ ID NO: 47) Cys Thr Ala Cys Arg Trp Lys Lys Ala (SEQ ID NO: 48) ThrAla Cys Arg Trp Lys Lys Ala Cys (SEQ ID NO: 49) Ala Cys Arg Trp Lys LysAla Cys Gln (SEQ ID NO: 50) Cys Arg Trp Lys Lys Ala Cys Gln Arg (SEQ IDNO: 51) Cys Thr Ala Cys Arg Trp Lys Lys Ala Cys (SEQ ID NO: 52) Thr AlaCys Arg Trp Lys Lys Ala Cys Gln (SEQ ID NO: 53) Ala Cys Arg Trp Lys LysAla Cys Gln Arg (SEQ ID NO: 54) Cys Thr Ala Cys Arg Trp Lys Lys Ala CysGln (SEQ ID NO: 55) Thr Ala Cys Arg Trp Lys Lys Ala Cys Gln Arg

The peptide of the present invention includes one consisting of not onlya part of the amino acid sequence of SEQ ID NO: 2 but also a part ofproteins of the present invention having an amino acid sequencesubstantially the same as SEQ ID NO: 2, subject that said peptide has acharacteristic of binding to HLA antigen and being recognized by CTLs.Thus, a modified peptide (hereinafter, the modified peptide may bereferred to as “variant peptide”) consisting of a part of the protein ofthe present invention having an amino acid sequence wherein the aminoacid sequence shown in SEQ ID NO: 2 is partly modified (by deletion,substitution and/or addition of amino acid(s)) also falls within thescope of the present invention, subject that said peptide has acharacteristic of binding to HLA antigen and being recognized by CTLs.Specifically, a variant peptide having an amino acid sequence wherein atleast one amino acid modification has been introduced into the aminoacid sequence of the peptide of the present invention which consists ofa partial amino acid sequence of the protein of the present invention,specifically the amino acid sequence of SEQ ID NO: 2, and being capableof binding to HLA antigen and recognized by CTLs falls within the scopeof the present invention.

The “modification” of amino acid residues means substitution, deletionand/or addition of amino acid including addition to the N- and/orC-terminus of peptide, preferably substitution of amino acid. When themodification involves amino acid substitution, the number or position ofamino acid to be substituted can be selected arbitrarily; however, it ispreferred that the substitution involves 1 to several amino acids sincethe tumor antigen peptides are generally about 8-14 amino acids inlength.

Variant peptides of the present invention are preferably 8 to 14 aminoacids in length. However, in the cases of HLA-DR, -DP, -DQ, peptidesconsist of 14 amino acids or more are known.

As mentioned above, motifs for antigen peptides that bind to an HLA andpresented are known in regard to certain HLA types, such as HLA-A1,-A0201, -A0204, -A0205, -A0206, -A0207, -A11, -A24, -A31, -A6801, -B7,-B8, -B2705, -B37, -Cw0401 and -Cw0602. Further, it is possible tosearch for peptide sequences that are expected to be able to bind to HLAantigen via internet. Thus, one can prepare the variant peptides aboveon the basis of these motifs and the like.

For example, as hereinbefore described, motifs of antigen peptidescapable of binding to HLA-A24 and being presented are known that, in the8-11 amino acid peptide, the amino acid at position 2 is tyrosine,phenylalanine, methionine or tryptophan, and the C terminal amino acidis phenylalanine, leucine, isoleucine, tryptophan or methionine (J.Immunol., 152: p 3913, 1994; Immunogenetics, 41: p 178, 1995; J.Immunol., 155: p 4307, 1994). As for HLA-A2, the motifs listed in Table1 above are known. Furthermore, there are published via internet certainpeptide sequences that are expected to be able to bind to HLA antigen.Accordingly, amino acids having a similar characteristic to thoseavailable for the motif above are acceptable. Thus, the presentinvention includes variant peptides comprising an amino acid sequencewherein an amino acid(s) at position(s) available for substitution inlight of motif (in the case of HLA-A24 and HLA-A2, position 2 andC-terminus) is substituted by other amino acid, preferably, an aminoacid expected to have binding activity as a result of internet search,and having an activity of binding to HLA and being recognized by CTLs.

More preferably, the present invention includes variant peptidescomprising an amino acid sequence wherein an amino acid(s) at thatposition(s) is substituted by any amino acid(s) known to be available inlight of motif and having the said activity. Thus, in the case of HLA-A24-restricted peptides, as shown in SEQ ID NO: 6-45, examples of variantpeptides include those having an amino acid sequence wherein the aminoacid at position 2 is substituted by tyrosine, phenylalanine, methionineor tryptophan, and/or the C terminal amino acid by phenylalanine,leucine, isoleucine, tryptophan or methionine, and having theaforementioned activity are included. Above all, a peptide wherein theamino acid at the second position is substituted by tyrosine is morepreferred.

The peptide of the present invention includes an epitope peptidecomprising the above-mentioned peptide of the present invention.

Recently, a peptide (“epitope peptide”) composed of multiple (plural)CTL epitopes (antigen peptides) linked together has been shown to induceCTLs efficiently For example, it has been reported that a peptide (about30-mer) wherein CTL epitopes each restricted to HLA-A2-, -A3, -A11, B53originated from tumor antigen protein PSA are ligated together inducedin vivo CTLs specific for respective CTL epitopes (Journal of Immunology1998, 161: 3186-3194).

In addition, a peptide (epitope peptide) wherein a CTL epitope and ahelper epitope are ligated has been shown to induce CTLs efficiently. Inthis context, “helper epitope” means a peptide capable of activating aCD4-positive T cells (Immunity., 1:751, 1994), and examples thereofinclude HBVc 128-140 of hepatitis B virus origin, TT947-967 of tetanustoxin origin, etc. CD4+ T cells activated by said helper epitope exertactivities including induction and maintenance of CTLs, and activationof effectors such as macrophages, etc, and hence are considered to beimportant in the immunological anti-tumor response. As a concreteexample of a peptide composed of a helper epitope and a CTL epitopelinked together, it is reported that a DNA (minigene) composed ofHBV-originated HLA-A2-restricted tumor antigen peptides (6 peptides),HLA-All-restricted tumor antigen peptides (3 peptides) and a helperepitope induced in vivo CTLs directed to the respective epitopesefficiently (Journal of Immunology 1999, 162: 3915-3925). Practically, apeptide wherein a CTL epitope (tumor antigen peptide corresponding toposition 280-288 of melanoma antigen gp 100) and a helper epitope(tetanus toxin-originated T helper epitope) are ligated has beensubjected to clinical test (Clinical Cancer Res., 2001, 7:3012-3024).

Accordingly, the peptide of the present invention also includes apeptide (epitope peptide) consisting of multiple epitopes including thepeptide of the present invention ligated therein and having an activityof inducing CTLs.

In this respect, the “epitope peptide” is defined as a peptide that is(a) a peptide consisting of two or more CTL epitopes (tumor antigenpeptides) ligated therein, or (b) a peptide consisting of a CTL epitopeand a helper epitope ligated therein, which is processed in anantigen-presenting cell to give a tumor antigen peptide, which tumorantigen peptide is then presented by said cell and induces CTLs.

When the epitope to be ligated to the peptide of the present inventionis a CTL epitope, examples of CTL epitopes usable include those derivedfrom the amino acid sequence shown in SEQ ID NO: 2 which are restrictedto HLA-A1, -A0201, -A0204, -A0205, -A0206, -A0207, -A11, -A24, -A31,-A6801, -B7, -B8, -B2705, -B37, -B55, -Cw0401, -Cw0602, and the like.CTL epitopes originated from other tumor antigen proteins are alsousable. Plural number of CTL epitopes can be linked together, and thelength of a CTL epitope may be about 8-14 amino acids based on theanalysis of antigen peptides binding to various HLA molecules(Immunogenetics, 41: 178, 1995).

When the epitope to be ligated to the peptide of the present inventionis a helper epitope, examples of helper epitopes usable include theaforementioned HBVc128-140 of hepatitis B virus origin, TT947-967 oftetanus toxin origin, etc. The helper epitope may be about 13-30 aminoacids, preferably, about 13-17 amino acids in length.

The peptide (epitope peptide) composed of multiple epitopes ligatedtherein can be prepared by aforementioned usual method for peptidesynthesis. It can also be prepared by a usual method for DNA synthesisand genetic engineering on the basis of sequence information of apolynucleotide encoding epitope peptide composed of multiple epitopesligated therein. That is, an intended epitope peptide wherein multipleepitopes are ligated can be prepared by inserting a polynucleotideencoding the polynucleotide into a known expression vector, transforminga host cell with the resultant recombinant expression vector, culturingthe transformants, and recovering the peptide from the culture. Theseprocesses can be conducted according to, for example, a method describedin a literature (Molecular Cloning, T. Maniatis et al., CSH Laboratory(1983), DNA Cloning, DM. Glover, IRL PRESS (1985)).

The so produced epitope peptide wherein multiple epitopes are ligatedcan be examined for the CTL-inducing activity in vitro by means of anassay as mentioned above, or in vivo by means of an assay described inWO02/47474 or Int J. Cancer: 100, 565-570 (2002) using a model animalfor human.

Also, the amino group of the N-terminal amino acid or the carboxyl groupof the C-terminal amino acid of the peptides of the present inventioncan be modified. The peptides undergone such modification also fallwithin the scope of the present invention.

Examples of a group for the modification of amino group of theN-terminal amino acid include 1 to 3 groups selected from C₁₋₆ alkylgroup, phenyl group, cycloalkyl group and acyl group. Acyl groupsspecifically includes C₁₋₆ alkanoyl group, C₁₋₆ alkanoyl groupsubstituted by phenyl group, carbonyl group substituted by C₅₋₇cycloalkyl group, C₁₋₆ alkylsulfonyl group, phenylsulfonyl group, C₂₋₆alkoxycarbonyl group, alkoxycarbonyl group substituted by phenyl group,carbonyl group substituted by C₆₋₇ cycloalkoxy group, phenoxycarbonylgroup, etc.

Examples of peptides modified at the carboxyl group of C-terminal aminoacid include esters and amides. Esters specifically include C₁₋₆ alkylesters, C₀₋₆ alkyl esters substituted by phenyl group, C₅₋₇ cycloalkylesters, etc. Amides specifically include amides, amides substituted byone or two C₁₋₆ alkyl groups, amides substituted by one or two C₀₋₆alkyl groups that are substituted by phenyl group, amides forming 5- to7-membered azacycloalkane inclusive of nitrogen atom of amide group,etc.

3) Polynucleotide of the Present Invention and Nucleic Acid Comprisingthe Polynucleotide

The nucleic acid (hereinafter, it may be referred to as “nucleic acid ofthe present invention”) contained in the inducer of CTL of the presentinvention comprises a polynucleotide (hereinafter, it may be referred toas “polynucleotide of the present invention”) encoding theaforementioned protein of the present invention.

The polynucleotide of the present invention can be cDNA or mRNA, cRNA orgenomic DNA of various cells or tissues originated from, for example,osteosarcoma or renal cancer, or synthetic DNA. It may be in eitherforms of single and double strands. Specifically, the polynucleotide ofthe present invention includes the followings:

(a) a polynucleotide comprising the base sequence shown in SEQ ID NO:1;

(b) a polynucleotide comprising the position 337-1878 of the basesequence shown in SEQ ID NO:1;

(c) a polynucleotide comprising a base sequence encoding the amino acidsequence shown in SEQ ID NO:2;

(d) a polynucleotide comprising the base sequence shown in SEQ

ID NO:3; and a polynucleotide comprising substantially the same basesequence as polynucleotides (a)-(d).

In this respect, the base sequence shown in SEQ ID NO:1 is registeredwith GenBank database under Accession No. AF263928, and encodes humanpapillomavirus binding factor (PBF, SEQ ID NO:2) described in Virology293, 103-117 (2002). The position 337-1878 is the open reading frame.The polynucleotide comprising the base sequence shown in SEQ ID NO: 3 isan analogous polynucleotide comprising the same base sequence portion(s)with the base sequence of SEQ ID NO: 1. Specifically, the partialsequence at position 1-1469 of the base sequence of SEQ ID NO:3 has 100%sequence identity with the partial sequence at position 704-1878 of thebase sequence of SEQ ID NO:1, except for the 294 by sequence that ispresent only in SEQ ID NO: 3.

Specifically, the above-defined polynucleotides (a)-(d), i.e., (a) apolynucleotide comprising the base sequence shown in SEQ ID NO:1; (b) apolynucleotide comprising the position 337-1878 of the base sequenceshown in SEQ ID NO:1; (c) a polynucleotide comprising a base sequenceencoding the amino acid sequence shown in SEQ ID NO:2; or (d) apolynucleotide comprising the base sequence shown in SEQ ID NO:3 includepolynucleotides consisting of a base sequence shown in any one of SEQ IDNO:1, position 337-1878 of SEQ ID NO:1 and SEQ ID NO:3, and apolynucleotide consisting of a base sequence encoding the amino acidsequence shown in SEQ ID NO:2. Further example includes a polynucleotideconsisting of a base sequence wherein additional base sequence is addedat the 5′- and/or 3′-terminus of the base sequence of SEQ ID NO:1,position 337-1878 of SEQ ID NO:1 or SEQ ID NO:3, or a base sequenceencoding the amino acid sequence of SEQ ID NO:2.

The polynucleotide is characterized by that the protein encoded by saidpolynucleotide has an activity of substantially the same quality as thatconsisting of the amino acid sequence shown in SEQ ID NO:2. The term “anactivity of substantially the same quality” means that a cell expressingthe protein encoded by the polynucleotide of the present invention has acharacteristic of being recognized by CTLs. Such activity and method ofdetermination are the same as described in “1) The Protein of thePresent Invention”.

A polynucleotide comprising the base sequence shown in SEQ ID NO: 1 or 3can be cloned by screening a cDNA library derived from, for example,osteosarcoma cell lines such as SaOS-2 using an appropriate portion ofthe base sequence disclosed in GenBank Accession No. AF263928 or the oneherein disclosed in SEQ ID NO: 1 or 3 as a probe for hybridization or aprimer for PCR. One ordinary skilled in the art can easily conduct thecloning according to the method described in Molecular Cloning 2nd Edt.Cold Spring Harbor Laboratory Press (1989), etc.

Examples of a polynucleotide comprising substantially the same basesequence as any one of polynucleotides (a) to (d) above specificallyinclude the followings: (e) a polynucleotide capable of hybridizing to acomplementary strand of any one of polynucleotides (a) to (d) understringent conditions, and being characterized by that a cell expressinga protein encoded by the polynucleotide is recognized by CTLs;

(f) a polynucleotide comprising a base sequence having at least 70%sequence identity with a polynucleotide set forth in any one of (a) to(d) above, and being characterized by that a cell expressing a proteinencoded by the polynucleotide is recognized by CTLs; and

(g) a polynucleotide encoding a protein comprising an amino acidsequence, wherein one or more amino acids are deleted, substitutedand/or added in the amino acid sequence encoded by any one ofpolynucleotides (a) to (d) above, and being characterized by that a cellexpressing the protein encoded by the polynucleotide is recognized byCTLs.

Preferred examples include a polynucleotide consisting of substantiallythe same base sequence as any one of polynucleotides (a) to (d) above.Examples of a polynucleotide consisting of substantially the same basesequence as any one of polynucleotides (a) to (d) above include thepolynucleotides (e′) to (g′) below.

(e′) a polynucleotide capable of hybridizing to a complementary strandof any one of polynucleotides (a) to (d) above under stringentconditions, and being characterized by that a cell expressing a proteinencoded by the polynucleotide is recognized by CTLs;

(f) a polynucleotide comprising a base sequence having at least 70%sequence identity with a polynucleotides set forth in any one of (a) to(d) above, and being characterized by that a cell expressing a proteinencoded by the polynucleotide is recognized by CTLs; and

(g′) a polynucleotide encoding a protein comprising an amino acidsequence, wherein one or more amino acids are deleted, substitutedand/or added in the protein encoded by any one of polynucleotides (a) to(d) above, and being characterized by that a cell expressing the proteinencoded by the polynucleotide is recognized by CTLs.

Examples of “a polynucleotide capable of hybridizing to a complementaryof any one of polynucleotides (a) to (d) above under stringentconditions” include polynucleotides comprising base sequences having atleast about 40%, preferably, about 60%, more preferably, about 70%,still more preferably about 80%, further more preferably about 90%, andmost preferably, about 95% sequence identity with the base sequence ofany one of polynucleotides (a) to (d) above, and specifically,polynucleotides consisting of partial sequences of any one ofpolynucleotides (a) to (d) above.

Hybridization can be conducted according to a method known per se or amethod equivalent thereto, for example, a method described in afundamental text “Molecular Cloning 2nd Edt. Cold Spring HarborLaboratory Press (1989)”, and the like. Also, it can be performed usinga commercially available library according to the instructions attachedthereto.

The “stringent conditions” herein used can be determined on the basis ofthe melting temperature (Tm) of nucleic acids forming a complex orbinding to probe as described in literatures (Berger and Kimmel, 1987,“Guide to Molecular Cloning Techniques Methods in Enzymology”, Vol. 152,Academic Press, San Diego Calif.; or “Molecular Cloning” 2nd Edt. ColdSpring Harbor Laboratory Press (1989)).

For example, hybridization can be carried out in a solution containing6×SSC (20×SSC means 333 mM sodium citrate, 333 mM NaCl), 0.5% SDS and50% formamide at 42° C., or in a solution containing 6×SSC (without 50%formamide) at 65° C.

Washing after hybridization can be conducted under a condition around“1×SSC, 0.1% SDS, 37° C.”. The complementary strand preferably remainsbound to the target sense strand when washed under such washingconditions. More stringent hybridization conditions may involve washingunder the conditions of around “0.5×SSC, 0.1% SDS, 42° C.” and stillmore stringent hybridization conditions involve washing conditions ofaround “0.1×SSC, 0.1% SDS, 65° C.”, although it is not limited thereto.

Examples of “a polynucleotide comprising a base sequence having at least70% sequence identity with a polynucleotides set forth in any one of (a)to (d) above” include polynucleotides comprising base sequences havingat least about 70%, preferably, about 80%, more preferably, about 90%,and most preferably, about 95% sequence identity with the base sequenceof any one of polynucleotides (a) to (d) above, and specifically,polynucleotides consisting of partial sequences of any one ofpolynucleotides (a) to (d) above.

The term “sequence identity” herein used refers to the identity andhomology between two polynucleotides. The “sequence identity” isdetermined by comparing two sequences aligned optimally over the regionscorresponding to the sequences to be compared. In this context, the bothpolynucleotides to be compared may have addition or deletion (e.g.,“gap”) in their sequences for optimum alignment. Such sequence identitycan be calculated by preparing alignment using, for example, Vector NTI,ClustalW algorithm (Nucleic Acid Res., 22 (22): 4673-4680 (1994)). Thesequence identity can be determined using software for sequenceanalysis, specifically, Vector NTI or GENETYX-MAC, or a sequencing toolprovided by a public database.

Polynucleotides having such sequence identity can be prepared accordingto the aforementioned hybridization method, conventional PCR reaction orreaction for modifying a polynucleotide (deletion, addition orsubstitution) hereinafter described.

Examples of “a polynucleotide encoding a protein comprising an aminoacid sequence, wherein one or more amino acids are deleted, substitutedand/or added in the protein encoded by any one of polynucleotides (a) to(d) above” include modified proteins produced artificially or allelevariants present in a living body.

In this respect, there is no limitations regarding the number orposition of modification (mutation) of amino acid as far as the activityof the protein of the present invention is maintained. Criteria based onwhich one can determine the number or position of amino acid residue tobe deleted, substituted and/or added without reducing the activity canbe obtained using a computer program well known in the art, such as DNAStar software. For example, the number of mutation would typically bewithin 10%, preferably 5% of the total amino acid residues. Furthermore,the amino acid used for substitution preferably has similarcharacteristics to the one to be substituted in view of retention ofstructure, which characteristics include polarity, charge, solubility,hydrophobicity, hydrophilicity, amphipathicity, etc. For instance, Ala,Val, Leu, Ile, Pro, Met, Phe and Tip are classified into nonpolar aminoacids; Gly, Ser, Thr, Cys, Tyr, Asn and Gln into non-charged aminoacids; Asp and Glu into acidic amino acids; and Lys, Arg and H is intobasic amino acids. One of ordinary skill in the art can select anappropriate amigo acid(s) falling within the same group on the basis ofthese criteria.

The polynucleotide encoding such protein variants may be prepared byvarious methods such as site-directed mutagenesis and PCR techniquedescribed in Molecular Cloning: A Laboratory Manual 2nd Edt. vols. 1-3,Cold Spring Harbor Laboratory Press (1989). It also can be prepared by aknown method such as Gapped duplex or Kunkel method using a commerciallyavailable kit

The polynucleotides of the present invention encode proteins having anactivity of substantially the same quality as a protein encoded by theamino acid sequence shown in SEQ ID NO:2. The term “activity ofsubstantially the same quality” refers to the characteristic future thatcells expressing a protein encoded by the polynucleotide of the presentinvention are recognized by CTLs. The said activity and the method ofdetermination are as described in “1) Proteins of the present invention”above.

The nucleic acid comprising the polynucleotide of the present inventionmay be in either forms of single and double strands. When thepolynucleotide of the present invention is double stranded, anexpression vector for expressing the protein of the present inventioncan be constructed by incorporating the above-mentioned polynucleotideinto an expression vector. Thus, the nucleic acid of the presentinvention encompasses an expression vector obtainable by inserting adouble strand polynucleotide of the present invention. Thus the presentinvention encompasses an expression vector constructed by inserting adouble strand polynucleotide of the present invention.

An adequate expression vector can be selected depending on the host tobe used, purposes, and the like, and include plasmids, phage vectors,virus vectors, etc.

When the host is Escherichia coli, examples of vector include plasmidvectors such as pUC118, pUC119, pBR322, pCR3, etc.; and phage vectorssuch as λZAPII, λgt11, etc. When the host is yeast, examples of vectorinclude pYES2, pYEUra3, etc. When the host is insect cells, vectorincludes pAcSGH is NT-A, etc. When the host is animal cells, examples ofinclude plasmid vectors such as pCEP4, pKCR, pCDM8, pGL2, pcDNA3.1,pRc/RSV, pRc/CMV, etc; and virus vectors such as retrovirus vector,adenovirus vector, adeno-associated virus vector, etc.

The expression vector may optionally contain a factor(s) such aspromoter capable of inducing expression, a gene encoding a signalsequence, a marker gene for selection, terminator, etc.

Furthermore, the expression vector may contain an additional sequencefor expressing the protein as a fusion protein with thioredoxin, Histag, GST (glutathione S-transferase), or the like, so as to facilitatethe isolation and purification. Vectors usable in such a case includeGST fusion protein vectors containing an appropriate promoter (lac, tac,trc, trp, CMV, SV40 early promoter, etc) that functions in host cells,such as pGEX4T; vectors containing Tag sequence (Myc, H is, etc) such aspcDNA3.1/Myc-His; and vectors capable of expressing a fusion proteinbetween thioredoxin and H is such as pET32a.

Transformed cells containing the vector of the present invention can beprepared by transformant host cells with an expression vector obtainedin the above.

Host cells usable herein include Escherichia coli, yeast, insect cellsand animal cells. Examples of Escherichia coli include strains of E.coli K-12 such as HB101, C600, JM109, DH5α and AD494 (DE3). Examples ofyeast include Saccharomyces cerevisiae. Examples of animal cells includeL929, BALB/c3T3, C127, CHO, COS, Vero, Hela and 293-EBNA cells. Examplesof insect cells include sf9.

Introduction of an expression vector into host cells can be done using aconventional method suited for the respective host cells above.Specifically, it can be done with calcium phosphate method, DEAE-dextranmethod, electroporation method, and a method using lipid for genetransfer (Lipofectamine Lipofectin; Gibco-BRL). Following theintroduction, the cells are cultured in a conventional medium containinga selection marker, whereby transformants containing the expressionvector can be selected.

The protein of the present invention can be produced by culturing thetransformed cells under appropriate conditions, the protein of thepresent invention can be produced. The resultant protein may be furtherisolated and purified according to standard biochemical procedures.Thus, purification procedures include salting out, ion exchangechromatography, absorption chromatography, affinity chromatography, gelfiltration chromatography, etc. When the protein of the presentinvention has been expressed as a fusion protein with thioredoxin, Histag, GST, or the like, as mentioned above, the said protein can beisolated and purified by appropriate purification procedures making useof the characteristics of such fusion protein or tags.

Nucleic acids comprising polynucleotides encoding the peptide of thepresent invention fall within the scope of the nucleic acid of thepresent invention.

The polynucleotide encoding a peptide of the present invention may be inthe form of DNA or RNA. The polynucleotide of the present invention canbe easily prepared on the basis of information about the amino acidsequence of the peptide or DNA encoding the same. Specifically, it canbe prepared by a conventional method such as DNA synthesis oramplification by PCR.

Specifically, the polynucleotide encoding a peptide of the presentinvention includes polynucleotides encoding the aforementioned epitopepeptides.

The nucleic acid comprising a polynucleotide encoding a peptide of thepresent invention may be in either form of single- or double-strand.When the polynucleotide of the present invention forms a double strand,an expression vector for expressing the peptide of the present invention(epitope peptide) can be constructed by incorporating theabove-mentioned polynucleotide into an expression vector.

The expression vector, host cell, a method for transforming a host cell,and the like herein used are similar to those described above.

4) Inducer of CTL Comprising as an Active Ingredient a Protein of thePresent Invention

The cell comprising the protein of the present invention has acharacteristic of being recognized by CTLs. That is, the protein of thepresent invention is an inducer of CTL. The so induced CTLs are capableof exhibiting anti-tumor effects through cytotoxic action or productionof lymphokines. Accordingly, the protein of the present inventions canbe used as an active ingredient of a medicine (cancer vaccine) fortreatment or prevention of tumor. The inducer of CTLs comprising aprotein of the present invention as an active ingredient exertstherapeutic or preventive effects when administered to a tumor patientin the following manner. The protein, when administered to a tumorpatient, is incorporated by antigen-presenting cells and intracellularlydegradated; the resultant tumor antigen peptides generated byintracellular degradation bind to HLA antigen to form complexes; thecomplexes are then presented on the surface of antigen-presenting cells;and CTLs specific for the complex efficiently proliferate in the bodyand destroy tumor cells. In this way, treatment or prevention of tumorsis achieved.

The inducer of CTL comprising as an active ingredient a protein of thepresent invention can be administered to any tumor patients who arepositive for PBF protein shown in SEQ ID NO: 2. Specifically, it can beused for prevention or treatment of all sorts of sarcomas such asosteosarcoma, or a cancer (tumor) such as renal cancer.

The inducer of CTL comprising as an active ingredient a protein of thepresent invention may be administered together with a pharmaceuticallyacceptable carrier, for example, an appropriate adjuvant, so that thecellular immunity can be established effectively

Examples of adjuvant applicable include those described in a literature(Clin. Microbiol. Rev., 7:277-289, 1994). Specifically, the followingsare contemplated: components derived from microorganisms or derivativethereof, cytokines, components derived from plants or derivativesthereof, components derived from marine organisms or derivativesthereof, mineral gels such as aluminium hydroxide, lysolecithin,surfactants such as Pluronic® polyols, polyanion, peptide, oil emulsion(emulsion preparation) and the like. In addition, liposomalpreparations, particulate preparations in which the ingredient is boundto beads having a diameter of several μm, preparations in which theingredient is attached to lipids, microsphere preparations, andmicrocapsules are also contemplated.

In this context, the “components derived from microorganisms orderivative thereof” can be specifically classified into (a) killedbacteria, (b) Cell Wall Skeleton (hereinafter, “CWS” derived frombacteria), and (c) particular components derived from microorganisms andderivatives thereof.

(a) Examples of killed bacteria include powdery hemolytic streptococcus(e.g., Picibanil®, Chugai Co., Ltd.), cocktail of killed bacteriumsuspension (e.g., Broncasma Berna®, Sanwa Kagaku Kenkyusho Co., Ltd) orkilled bacteria of Mycobacterium tuberculosis, and the like.

(b) Examples of CWS derived from bacteria include CWS fromMicrobacterium (e.g., Mycobacterium bovis CWS), CWS from Nocardia (e.g.,Nocardia rubra CWS), Corynebacterium CWS, etc.

(c) Examples of particular components derived from microorganisms andderivatives thereof include microorganism-derived polysaccharides suchas polysaccharides from Mycobacterium tuberculosis (e.g., Ancer®, ZeriaPharmaceutical Co., Ltd.); polysaccharides from Basidiomycetes(Lentinan®, Ajinomoto, Co., Ltd.; Krestin®, Sankyo, Co., Ltd.;Basidiomycetes, Coriolus versicolor (Fr) Quel); muramyl dipeptide (MDP)associated compounds; lipopolysaccharides (LPS); lipid A (MPL)associated compounds; glycolipids trehalose dimycolate (TDM); bacteriumDNA (e.g., CpG oligonucleotide); and derivatives thereof.

These microorganism-derived components and derivatives thereof can beavailable from commercial source or can be produced and isolatedaccording to a method described in known literatures (e.g., Cancer Res.,33, 2187-2195 (1973); J. Natl. Cancer Inst., 48, 831-835 (1972), J.Bacteriol., 94, 1736-1745 (1967); Gann, 69, 619-626 (1978), J.Bacteriol., 92, 869-879 (1966) or J. Natl. Cancer Inst., 52, 95-101(1974)).

The term “cytokines”, for example, refers to IFN-α, IL-12, GM-CSF, IL-2,IFN-γ, IL-18 or IL-15. These cytokines may be a product of nature orgenetic engineering. When a cytokine(s) is commercially available, onecan pursue and use the same. Alternatively, cytokines can be preparedrecombinantly by cloning a desired gene in a conventional manner on thebasis of respective base sequences registered with database such asGenBank, EMBL or DDBJ, ligating the gene into an appropriate expressionvector, transforming host cells with the resultant recombinantexpression vector, and allowing the cells to express and produce theintended cytokine.

Examples of the “components derived from plants or derivatives thereof”include saponin-derived component Quil A (Accurate Chemical & ScientificCorp), QS-21 (Aquila Biopharmaceuticals Inc.), or glycyrrhizin(SIGMA-ALDRICH, etc.).

Examples of the “components derived from marine organisms or derivativesthereof” include sponge-derived glycolipid α-galactosylceramide.

Examples of oil emulsion (emulsion preparation) include emulsionpreparations of water-in-oil type (w/o), oil-in-water type (o/w) andwater-in-oil-in-water type (w/o/w). In the water-in-oil type (w/o)emulsion preparation, an active ingredient is dispersed in water as thedisperse phase. In the oil-in-water type (o/w) emulsion preparation, anactive ingredient is dispersed in water as the disperse medium. Further,in the water-in-oil-in-water type (w/o/w) emulsion preparation, anactive ingredient is dispersed in water as the most internal phase. Suchemulsion preparations can be produced in accordance with the teachingin, for example, JP-A-8-985, JP-A-9-122476, or the like.

The “liposomal preparations” refer to microparticles wherein an activeingredient in water phase or membrane is encapsulated with liposomes oflipid bilayer structure. Major lipids for preparation of liposomesinclude phosphatidyl choline, sphingomyelin, etc. Dicetyl phosphate,phosphatidic acid, phosphatidyl serine or the like that confers chargemay also be added for stabilization of liposomes. The method ofproducing liposomes include ultrasonic method, ethanol injection method,ether injection method, reverse phase evaporation method, French pressextraction method, and the like.

The “microsphere preparations” refer to microparticles composed of ahomogeneous polymer matrix wherein an active ingredient is dispersed insaid matrix. The matrix can be composed of a biodegradable polymer suchas albumin, gelatin, chitin, chitosan, starch, polylactic acid,polyalkyl cyanoacrylate, and the like. The microsphere preparations canbe prepared by any of known methods without limitation, including thosedescribed in literatures (Eur. J. Pharm. Biopharm. 50:129-146, 2000;Dev. Biol. Stand. 92:63-78, 1998; Pharm. Biotechnol. 10:1-43, 1997,etc.).

The “microcapsule preparations” refer to microparticles containing anactive ingredient as a core substance which is enveloped with a film.The coating material used for film includes a film-forming polymer suchas carboxymethylcellulose, cellulose acetate phthalate, ethyl cellulose,gelatin, gelatin/acacia, nitrocellulose, polyvinyl alcohol,hydroxypropyl cellulose, and the like. The microcapsule preparations canbe prepared by coacervation method, surface polymerization, and thelike.

Administration may be achieved, for example, intradermally,subcutaneously, intramuscularly, or intravenously. Although the dosageof the protein of the present invention in the formulation to beadministered may be adjusted as appropriate depending on, for example,the disease to be treated, the age and the body weight of the patient,it is usually within the range of 0.0001-1000 mg, preferably, 0.001-100mg, more preferably 0.01-10 mg, which can be administered once in everyseveral days to every several months.

5) Inducer of CTL Comprising as an Active Ingredient a Peptide of thePresent Invention

The peptide of the present invention is an inducer of CTL having anactivity of inducing CTLs. The so induced CTLs can exert the anti-tumoreffects through cytotoxic action or production of lymphokines.Accordingly, the peptide of the present invention can be used as anactive ingredient of a medicine for treatment or prevention of tumor.When an inducer of CTL comprising as an active ingredient the peptide ofthe present invention is administered to a tumor patient, the peptide ofthe present invention is presented to an HLA antigen inantigen-presenting cells. Then, CTLs specific for the presented bindingcomplex between the HLA antigen and the peptide of the present inventionproliferate, which in turn destroy tumor cells. In this way, thetreatment or prevention of tumors in a patient can be achieved.

The inducer of CTL comprising as an active ingredient a peptide of thepresent invention can be administered to any tumor patients who arepositive for PBF protein shown in SEQ ID NO: 2. Specifically, it can beused for prevention or treatment of all sorts of sarcomas such asosteosarcoma, or a cancer (tumor) such as renal cancer.

The inducer of the present invention may comprise a single CTL epitope(peptide of the present invention) or a epitope peptide wherein the saidpeptide is ligated with other peptide(s) (CTL epitope or helper epitope)as an active ingredient. Recently, an epitope peptide composed ofmultiple (plural) CTL epitopes (antigen peptides) linked together hasbeen shown to have an activity of inducing CTLs efficiently For example,it has been reported that about 30-mer epitope peptide wherein CTLepitopes restricted to HLA-A2-, -A3,- -A11 or B53 originated from tumorantigen protein PSA are ligated induced CTLs specific for respective CTLepitopes (Journal of Immunology 1998, 161: 3186-3194). In addition, ithas been reported that an epitope peptide wherein a CTL epitope(s) and ahelper epitope(s) are ligated can induce CTLs efficiently. When thepeptide of the present invention is administered in the form of epitopepeptide, the said peptide is incorporated by antigen-presenting cells;respective antigen peptides generated by intracellular degradation bindto an HLA antigen to form complexes; the complexes are presented on thesurface of antigen-presenting cells in high density; CTLs specific forthe complexes efficiently proliferate in the body, and destroy tumorcells. In this way, treatment or prevention of tumors is achieved.

The inducer of CTL comprising as an active ingredient a protein of thepresent invention may be administered together with a pharmaceuticallyacceptable carrier, for example, an appropriate adjuvant, so that thecellular immunity can be established effectively

Examples of adjuvant applicable include those described in a literature(Clin. Microbiol. Rev., 7:277-289, 1994). Specifically, the followingsare contemplated: components derived from microorganisms or a derivativethereof, cytokines, components derived from plants or derivativesthereof, components derived from marine organisms or derivativesthereof, mineral gels such as aluminium hydroxide, surfactants such aslysolecithin, Pluronic® polyols, polyanion, peptide, oil emulsion(emulsion preparation) and the like. In addition, liposomalpreparations, particulate preparations in which the ingredient is boundto beads having a diameter of several μm, preparations in which theingredient is attached to lipids, microsphere preparations, andmicrocapsules are also contemplated. Concrete examples of theseadjuvants are the same as those descried in the above “4) CTL inducerscomprising a protein of the present invention”.

Administration may be achieved, for example, intradermally,subcutaneously, intramuscularly, or intravenously. Although the dosageof the protein of the present invention in the formulation to beadministered may be adjusted as appropriate depending on, for example,the disease to be treated, the age and the body weight of the patient,it is usually within the range of 0.0001-1000 mg, preferably 0.001-1000mg, more preferably 0.1-10 mg, which can be administered once in everyseveral days to every several months.

6) Inducer of CTL Comprising as an Active Ingredient a Nucleic Acid ofthe Present Invention

The cells expressing the nucleic acid of the present invention has acharacteristic of being recognized by CTLs. Accordingly, the nucleicacid of the present invention is an inducer of CTLs. The so induced CTLscan exert the anti-tumor effects through cytotoxic action or productionof lymphokines. The nucleic acid of the present invention therefore canbe used as an active ingredient of a medicine for treatment orprevention of tumor. The inducer comprising as an active ingredient thenucleic acid of the present invention, when administered, can exerttherapeutic or preventive effects on tumors through the expression ofthe nucleic acid.

For example, the nucleic acid of the present invention incorporated intoan expression vector is administered to a tumor patient in the followingmanner, tumor antigen proteins are highly expressed inantigen-presenting cells. Thereafter, tumor antigen peptides generatedby intracellular degradation form complexes with HLA antigen; thecomplexes are then presented on the surface of antigen-presenting cellsin high density; and tumor-specific CTLs proliferate in the bodyefficiently and destroy tumor cells. In this way, treatment orprevention of tumors is achieved.

The inducer of CTL comprising as an active ingredient a nucleic acid ofthe present invention can be administered to any tumor patients who arepositive for PBF gene shown in SEQ ID NO: 1 and for PBF that is anexpression product of said gene. Specifically, it can be used forprevention or treatment of all sorts of sarcomas such as osteosarcoma,or a cancer such as renal cancer

Administration and introduction of the nucleic acid of the presentinvention into cells may be achieved using viral vectors or according toany one of other procedures (Nikkei-Science, April, 1994, pp. 20-45;Gekkan-Yakuji, 36(1), 23-48 (1994); Jikken-Igaku-Zokan, 12(15), 1994,and references cited therein).

Examples of a method for introduction with viral vectors comprisesincorporating DNA of the present invention into DNA or RNA virus such asretrovirus, adenovirus, adeno-associated virus, herpesvirus, vacciniavirus, poxvirus, poliovirus, or Sindbis virus, and introducing intocells. Above all, a method that uses retrovirus, adenovirus,adeno-associated virus, or vaccinia virus is particularly preferred.

Examples of other methods include a method wherein an expression plasmidis directly injected intramuscularly (DNA vaccination), liposome method,Lipofectin method, microinjection, calcium phosphate method andelectroporation. DNA vaccination and liposome method are particularlypreferred.

Regarding method to make a nucleic acid of the present invention act asa medicament in practice, there are in vivo method wherein the nucleicacid is directly introduced into the body, and ex vivo method whereinthe nucleic acid is introduced extracorporeally into a certain cellsremoved from human, and the cells are reintroduced into the body(Nikkei-Science, April, 1994, pp. 20-45; Gekkan-Yakuji, 36(1), 23-48(1994); Jikkenn-Igaku-Zokan, 12(15), 1994; and references citedtherein). An in vivo method is more preferred.

In case of in vivo method, the administration can be effected throughany appropriate routes depending on the disease and symptom to betreated and other factors. For example, it may be administered viaintravenous, intraarterial, subcutaneous, intracutaneous, intramuscularroute, or the like. When the administration is carried out by in vivomethod, the compositions may be administered in various forms such assolution, and are typically formulated, for example, in the form ofinjection containing the nucleic acid of the present invention as anactive ingredient, to which conventional carriers may also be added, ifnecessary. As for the liposomes or membrane-fused liposomes (such asSendai virus (HVJ)-liposomes) containing the nucleic acid of the presentinvention, they may be in the form of liposomal formulation such assuspension, frozen drug, centrifugally-concentrated frozen drug, or thelike.

Although the dosage of the nucleic acid of the present invention in theformulation to be administered may be adjusted as appropriate dependingon, for example, the disease to be treated, the age and the body weightof the patient, it is usually, as the amount of nucleotide in thenucleic acid, within the range of 0.0001-100 mg, preferably, 0.001-10mg, which can be administered once in every several days to everyseveral months.

Recently, a polynucleotide encoding an epitope peptide wherein multiple(plural) CTL epitopes (antigen peptides) are ligated or wherein a CTLepitope(s) and a helper epitope(s) are ligated has been shown to induceCTLs in vivo efficiently For example, it is reported that a DNA(minigene) encoding an epitope peptide wherein HBV-originatedHLA-A2-restricted tumor antigen peptides (6 peptides),HLA-A11-restricted tumor antigen peptides (3 peptides) and a helperepitope are ligated induced in vivo CTLs directed to the respectiveepitopes efficiently (Journal of Immunology 1999, 162: 3915-3925).

Accordingly, a polynucleotide prepared by ligating one or morepolynucleotides encoding the peptide of the present invention,optionally in association with another polynucleotide(s) encodingdifferent peptide(s) can be used as an active ingredient afterintroducing into an appropriate expression vector. Such an inducer ofCTL may be applied to the same administration manner or form thatdescribed above.

7) Antigen-Presenting Cells of the Present Invention

The tumor antigen protein, peptide and nucleic acid of the presentinvention may also be used in vitro for treatment of tumor patients asfollows. That is, the antigen-presenting cells can be prepared bybringing a cell having antigen-presenting ability into contact with anyone of proteins, peptides and nucleic acids of the present invention invitro. Specifically, the present invention provides anantigen-presenting cell presenting a complex between an HLA antigen andany one of proteins, peptides and nucleic acids of the presentinvention, which cell has been prepared by bringing a cell havingantigen-presenting ability isolated from a tumor patient with any one ofproteins, peptides and nucleic acids of the present invention in vitro,and a method for preparing the same.

In this context, the “cells having antigen-presenting ability” are notlimited to particular cells and may be any cells that express on thesurface an HLA antigen capable of presenting the peptide of the presentinvention; however, dendritic cells known to have especially highantigen-presenting ability are preferred.

Further, the substance used for preparing the antigen-presenting cellsof the present invention from cells having antigen-presenting abilitymay be any of proteins, peptides and nucleic acids of the presentinvention.

The antigen-presenting cells of the present invention can be prepared byisolating from a tumor patient cells having antigen-presenting ability,pulsing the cells in vitro with a protein or peptide of the presentinvention, and allowing the cells to present a complex between an HLAantigen and a peptide of the present invention (Cancer Immunol.Immunother., 46: 82, 1998; J. Immunol. 158: p 1796, 1997; Cancer Res.,59:1184, 1999). When dendritic cells are used, antigen-presenting cellsof the present invention may be prepared, for example, by isolatinglymphocytes from peripheral blood of a tumor patient using Ficollmethod, removing non-adherent cells, incubating the adherent cells inthe presence of GM-CSF and IL-4 to induce dendritic cells, andincubating and pulsing said dendritic cells with a protein or a peptideof the present invention.

When antigen-presenting cells of the present invention are prepared byintroducing a nucleic acid of the present invention into theaforementioned cells having an antigen-presenting ability, said nucleicacid may be in the form of DNA or RNA. In particular, DNA may be usedaccording to the teaching in Cancer Res., 56:5672, 1996 or J. Immunol.,161:p 5607, 1998, and RNA according to the teaching in J. Exp. Med.,184:p 465, 1996, for example.

The aforementioned antigen-presenting cells can be used as an activeingredient of an inducer of CTL. The inducer of CTL comprising as anactive ingredient the said antigen-presenting cells preferably containsphysiological saline, phosphate buffered saline (PBS), medium, or thelike to stably maintain the antigen-presenting cells. It may beadministered, for example, intravenously, subcutaneously, orintradermally. Reintroduction of an inducer of CTL comprising as anactive ingredient antigen-presenting cells as an active ingredient intoa PBF-positive patient brings about efficient induction of specific CTLsin the body of said patient, and, as a result, treatment of tumor.

8) CTLs of the Present Invention

The protein, peptide and the nucleic acid of the present invention canbe used in vitro in treatment of tumor patients in the following manner.That is, any one of protein, peptide and the nucleic acid of the presentinvention can be used to induce CTLs in vitro by bringing the same intocontact with peripheral lymphocytes. Specifically, the present inventionprovides a CTL induced by bringing peripheral blood lymphocytes from atumor patient into contact in vitro with any one of protein, peptide andnucleic acid of the present invention and the method of induction ofCTL.

For melanomas, therapeutic effect has been observed in adoptiveimmunotherapy wherein tumor-infiltrating T cells are removed from apatient, cultured ex vivo in large quantities and returned into the samepatient (J. Natl. Cancer. Inst., 86: 1159, 1994). Further, in mousemelanoma, suppression of metastasis has been observed in treatmentwherein splenocytes were stimulated with tumor antigen peptide TRP-2 invitro thereby making CTLs specific for the tumor antigen peptide toproliferate, and the CTLs are administered to a melanoma-grafted mouse(J. Exp. Med., 185:453, 1997). This resulted from in vitro proliferationof CTLs that specifically recognize the complex between an HLA antigenof antigen-presenting cells and the tumor antigen peptide. Accordingly,a therapeutic method comprising stimulating in vitro peripheral bloodlymphocytes from a patient with a protein, peptide or nucleic acid ofthe present invention to proliferate tumor-specific CTLs, and returningthe CTLs into the patient is believed to be effective.

The CTLs can be used as an active ingredient of a therapeutic orpreventive agent for tumor. Said therapeutic or preventive agentpreferably contains physiological saline, phosphate buffered saline(PBS), medium, or the like to stably maintain CTLs. It may beadministered, for example, intravenously, subcutaneously orintradermally. By returning a therapeutic or preventive agent comprisingas an active ingredient the CTLs into a patient positive for PBF of thepresent invention, the cytotoxic action of CTLs is enhanced in the bodyof patient and whereby the tumor cells are killed and treatment of tumoris achieved.

9) Antibody Against the Peptide of the Present Invention

The present invention provides antibody capable of specifically bindingto the peptide of the present invention. The antibody of the presentinvention may be in the form of, although it is not limited to,polyclonal or monoclonal antibody raised against a peptide of thepresent invention. Although there are no limitations regarding theantibody of the present invention subject that it specifically binds tothe peptide of the present invention, concrete examples includeantibodies that specifically bind to a tumor antigen peptide consistingof an amino acid sequence of any one of those described in SEQ IDNO:6-55.

Preparation of Antibodies is Well Known in the Art and the Antibodies ofthe present invention can be prepared according to any one of theseconventional methods (Current protocols in Molecular Biology edit.Ausubel et al. (1987) Publish. John Wiley and Sons. Section 11.12-11.13,Antibodies; A Laboratory Manual, Lane, H, D. et al., ed., Cold SpringHarber Laboratory Press, New York 1989).

Specifically, when the antibodies of the present invention arepolyclonal, they can be obtained by immunizing non-human animal such asrabbit using a peptide of the present invention (e.g., a tumor antigenpeptide consisting of amino acid sequence shown in any one of SEQ ID NO:6-55) as an antigen, and recovering the antibodies from serum of theimmunized animal in a conventional manner. In the case of monoclonalantibodies, they can be obtained by immunizing non-human animal such asmouse with a peptide of the present invention (e.g., a tumor antigenpeptide consisting of amino acid sequence shown in any one of SEQ ID NO:6-55), subjecting the resultant splenocytes to cell fusion with myelomacells, and recovering antibodies from hybridoma cells (Current protocolsin Molecular Biology edit. Ausubel et al. (1987) Publish. John Wiley andSons. Section 11.4-11.11).

The antibodies against the peptide of the present invention can also beproduced while enhancing the immunological response using differentadjuvants depending on the host. Examples of adjuvants include Freundadjuvants; mineral gels such as aluminium hydroxide; surfactants such aslysolecithin, Pluronic® polyol, polyanion, peptide, oil emulsion,keyhole limpet hemocyanin and dinitorophenol; human adjuvants such asBCG (Bacille de Calmette-Guerin) or Corynebacterium, etc.

As mentioned above, antibodies that recognize a peptide of the presentinvention and antibodies that neutralize activity thereof may easily beprepared by immunizing an animal in a conventional manner. Theantibodies may be used in affinity chromatography, immunologicaldiagnostic method, and the like. Immunological diagnostic method may beselected as appropriate from immunoblotting, radioimmunoassay (RIA),enzyme-linked immunosorbent assay (ELISA), a fluorescent or luminescentassay, and the like. The immunological diagnostic method would beeffective for diagnosing cancers expressing the PBF gene of the presentinvention, such as sarcomas and renal cancers.

10) Tumor Marker 10-1) Tumor Marker Related to Polynucleotide of thePresent Invention

The present inventors have found that PBF gene defined in SEQ ID NO:1 ishighly expressed specifically in sarcomas and renal cancers compared tonormal cells. Accordingly, the presence or absence of a tumor especiallysarcoma or renal and the degree thereof can be specifically detected byexamining the presence or extent of expression of the gene, which inturn allows the diagnostic method of such diseases.

Accordingly, the polynucleotide of the present invention is useful as atool (tumor marker) for diagnosing whether or not a test subject issuffering from a tumor, or degree of the disease, wherein thepresence/absence or extent of expression of PBF gene above in a testsubject is detected using the polynucleotide.

The tumor marker of the present invention is characterized by that itcomprises at least 15 contiguous nucleotides in the base sequence ofaforementioned polynucleotide of the present invention, which encodes aprotein comprising the same or substantially the same amino acidsequence as that shown in SEQ ID NO: 2.

Specifically, the tumor marker of the present invention includes apolynucleotide and/or a complementary polynucleotide thereof, whichpolynucleotide comprises at least 15 contiguous nucleotides in the basesequence of SEQ ID NO 1 or SEQ ID NO: 3.

In this context, the term “complementary polynucleotide (complementarystrand, reverse strand (antisense strand)” refers to a polynucleotidewhich is in complementary relation in regard to bases (e.g., A:T, G:C)in the full- or partial-sequence (hereinafter, said sequence may bereferred to as “positive strand (sense strand)” for convenience) of thepolynucleotide consisting of the base sequence shown in SEQ ID NO: 1 or3, which partial sequence comprises at least 15 contiguous bases. The“complementary strand” includes both a sequence forming completelycomplementary strands with the base sequence of target positive strandand a sequence being complementary enough to hybridize with the basesequence of target positive strand under stringent conditions. Thestringent conditions herein used can be determined on the basis of themelting temperature (Tm) of nucleic acids forming a complex or bindingto probe as described in literatures (Berger and Kimmel, 1987, “Guide toMolecular Cloning Techniques Methods in Enzymology”, Vol. 152, AcademicPress, San Diego Calif.). For example, washing after hybridization canbe conducted under a condition around “1×SSC, 0.1% SDS, 37° C. in usual.The complementary strand preferably remains bound to the target sensestrand when washed under such washing conditions. More stringenthybridization conditions may involve washing under the conditions ofaround “0.5×SSC, 0.1% SDS, 42° C.” and still more stringenthybridization conditions involve washing conditions of around “0.1×SSC,0.1% SDS, 65° C.”, although it is not limited thereto. Suchcomplementary strand include specifically, for example, a strandconsisting of a base sequence having complete complementarity, ahomology of at least 90%, or preferably at least 95% in relation to thebase sequence of a target positive strand.

The polynucleotide of the positive strand may include not only thosecomprising the full or a partial sequence of the base sequence shown inSEQ ID NO: 1 or 3, but also those being in a complementary relation withthe base sequence of above-mentioned complementary strand.

Furthermore, the above-mentioned polynucleotides of positive or negative(reverse) strands can be used as a tumor marker in the form of singlestrand as well as double strand.

As a concrete example, the tumor marker of the present invention may bea polynucleotide consisting of a base sequence (full sequence) shown inSEQ ID NO: 1 or 3, or a polynucleotide comprising a complementary strandthereof. It may be a polynucleotide consisting of a partial sequence ofthe above-mentioned full sequence or its complementary sequence as longas said polynucleotide selectively (specifically) recognizes the gene ofthe present invention or a polynucleotide originated therefrom. Thepartial sequence includes a polynucleotide comprising a sequencecomprising at least contiguous 15 bases selected arbitrarily from thebase sequence of the aforementioned full sequence or a complementarysequence thereof.

The term “selectively (specifically) recognizes” used herein means that,in the case of Northern blotting, the PBF gene or a polynucleotidederived therefrom of the present invention can be specifically detected,and in the case of RT-PCR method, the PBF gene or a polynucleotidederived therefrom of the present invention can be specificallygenerated. However, the above definition is not restrictive and anycriteria can be used as long as one ordinary skilled in the art candetermine that the detected substances or products above are originatedfrom PBF gene.

For example, the tumor marker of the present invention can be designedon the basis of the base sequence shown in SEQ ID NO: 1 or 3 by means ofprimer 3 or a vector NTI (Infomax). Specifically, a candidate sequencefor primer or probe, or a sequence at least comprising said sequence asa partial sequence can be used as a primer or probe, which candidatesequence is obtainable by subjecting the base sequence of the gene ofthe present invention to primer 3 or vector NTI software. Example ofsuch a tumor marker of the present invention includes a primer shown inSEQ ID NO: 4 or 5.

The tumor marker of the present invention may be at least 15 bases inlength; however, depending on the use of the marker, the length can beappropriately selected and designated.

In the present invention, the detection (diagnostic method) of tumors isconducted by assessing the presence/absence or the level (amount ofproducts) of expression of PBF gene in a biopsy tissue especially samplefrom sarcoma or renal cancer of a patient suspected to be affected bytumor. In this case, the aforementioned tumor marker of the presentinvention may serve as a primer that specifically recognizes and bringsabout proliferation of an RNA resulted from the expression of PBF geneor a polynucleotide derived therefrom, or as a probe for detectingspecifically said RNA or a polypeptide derived therefrom.

The tumor makers of the present invention may include, when used as aprimer in the detection of tumors, those comprising a base sequence of,in general, 15-100 bp, preferably 15-50 bp, more preferably 15-35 by inlength. When used as a detection probe, the tumor markers may, ingeneral, comprise a base sequence of from 15 by to 1 kp, preferably from100 by to 1 kb.

The tumor marker of the present invention can be used as a primer or aprobe in a conventional manner in any of known methods for detectingspecifically a particular gene such as Northern blotting, RT-PCR, insitu hybridization, and the like.

The tumor marker of the present invention is useful in the diagnosticmethod or detection of tumor, that is, diagnostic method ofpresence/absence or pathology, or the extent thereof. Specifically,diagnostic method with the tumor marker can be conducted by comparingthe expression level of PBF gene in biopsy tissue suspected to beaffected by tumor of a test subject and that in a similar tissue of anormal subject. In this case, the “difference in expression level”refers not only the presence/absence of expression, but also theexpression differential is at least 2-fold, preferably 3-fold, whenexpression is observed in tissues of both groups. Specifically, sincethe expression of PBF gene is induced in tumors, a test subject issuspected to be affected by tumor, if the expression in a tissue sampleof the test subject is at least 2-fold, preferably 3-fold highercompared to that in the corresponding tissue of a normal subject.

10-2) Tumor Marker Related to Antibody of the Present Invention

The present invention provides an antibody capable of specificallyrecognizing a protein of the present invention or partial peptidesthereof (including the peptides of the present invention) as a tumormarker. More specifically, the present invention provides a tumor markercomprising an antibody capable of specifically recognizing a protein ofthe present invention consisting of the amino acid sequence shown in SEQID NO: 2 or partial peptides thereof (including the peptides of thepresent invention) as a tumor marker.

The present inventors have found that the PBF gene of the presentinvention is highly expressed in diverse sarcomas and renal cancers.Accordingly, the presence/absence of the tumor above (sarcoma, renalcancer, etc.) or the extent thereof can be detected specifically bydetecting the presence/absence of expression products of said gene orextent of expression, whereby such diseases can diagnosed.

Accordingly, the antibody above is useful as a tool (tumor marker) fordiagnosing whether or not a test subject is suffering from a tumor, ordegree of the disease, wherein the presence/absence or extent ofexpression of the protein above in a test subject is detected using theantibody.

The antibody of the present invention is not restricted to any form andincludes polyclonal or monoclonal antibody raised against a protein ofthe present invention as an immunogen, specifically, PBF proteinconsisting of the amino acid sequence shown in SEQ ID NO: 2. Theantibody of the present invention also includes those having ability tobind to polypeptides consisting of generally 8, preferably 15, morepreferably 22 contiguous amino acids in the protein of the presentinvention.

Preparation of Antibodies is Well Known in the Art and the Antibodies ofthe present invention can be prepared according to any one of theseconventional methods (Current protocols in Molecular Biology, Chapter11.12˜11.13 (2000))

Specifically, when the antibodies of the present invention arepolyclonal, they can be obtained by immunizing non-human animal such asrabbit with a protein of the present invention that has been expressedin E. coli and purified in a conventional manner, or a syntheticoligopeptide comprising a partial amino acid sequence of said protein ofthe present invention, and recovering the antibodies from serum of theimmunized animal in a conventional manner. In the case of monoclonalantibodies, they can be obtained by immunizing non-human animal such asmouse with a protein of the present invention that has been expressed inE. coli and purified in a conventional manner, or a syntheticoligopeptide comprising a partial amino acid sequence of said protein ofthe present invention, subjecting the resultant splenocytes to cellfusion with myeloma cells, and recovering antibodies from hybridomacells (Current protocols in Molecular Biology edit. Ausubel et al.(1987) Publish. John Wiley and Sons. Section 11.4-11.11).

The protein of the present invention used as an immunogen in thepreparation of antibody of the present invention (specifically, PBFprotein consisting of amino acid sequence shown in SEQ ID NO:2) isobtainable by processes comprising cloning of a DNA on the basis ofinformation herein provided (SEQ ID NO: 1, 3), construction of plasmid,transfection into a host cell, incubation of host cells, and recovery ofprotein from cultured medium. These processes can be conducted by anyone of methods known to those skilled in the art or described in aliterature (Molecular Cloning, T. Maniatis et al., CSH Laboratory(1983), DNA Cloning, DM. Glover, IRL PRESS (1985)), and the like. Thepartial peptides of the protein of the present invention can also beproduced by a usual method for chemical synthesis (peptide synthesis) onthe basis of information regarding amino acid sequence (SEQ ID NO:2)herein provided. The detail thereof is described above in 1) to 3).

The antibody of the present invention may be the one obtainable using anoligopeptide comprising a partial amino acid sequence of the protein ofthe present invention. An oligo(poly)peptides used in the preparation ofantibodies does not needed to possess a functional biological activity;however, it is desired that such a peptide possesses a similarimmunogenicity to the protein of the present invention. Preferredexamples include oligo(poly)nucleotides having said immunogenicity andcomprising at least 8, preferably 15, more preferably 20 contiguousamino acids in the amino acid sequence of the protein of the presentinvention.

The production of antibodies against an oligo(poly)peptide can also becarried out while administering to the host various adjuvants so as toenhance the immunoreactivity. Such adjuvants (but are not limitedthereto) include Freund adjuvants; mineral gels such as aluminiumhydroxide; surfactants such as lysolecithin, Pluronic® polyol,polyanion, peptide, oil emulsion, keyhole limpet hemocyanin anddinitorophenol; human adjuvants such as BCG (Bacille de Calmette-Guerin)or Corynebacterium, etc.

The antibody of the present invention has a characteristic ofspecifically binding to PBF protein, and hence makes it possible todetect specifically the above-mentioned protein expressed in tissues ofa test subject. Thus, the said antibody is useful as a probe fordetecting the presence/absence of expression of PBF in tissues of a testsubject.

Specifically, PBF can be detected by obtaining a sample from a tissue orblood suspected to be affected by biopsy or the like, preparing aprotein therefrom in a conventional manner, and conducting detection bya known method such as Western blotting, ELISA, or the like using theantibody as a probe.

Diagnostic method of tumors may be accomplished by determining thedifference in the quantity of PBF proteins in a tissue of test subjectand that in the corresponding normal tissue. In this case, thequantitative difference of protein may involve the cases where theprotein is either present or absent, and the amount of protein differsat least by 2-fold, preferably 3-fold. Specifically, since theexpression of PBF gene is induced in tumors (sarcoma, renal cancer), atest subject is suspected to be affected by tumor, if a tissue of saidtest subject contains the expression product (PBF) of the gene and thequantity of PBF therein is at least 2-fold preferably 3-fold larger thanthat in the normal tissue.

10-3) Tumor Marker Related to Protein or Peptide of the PresentInvention

The present invention provides, as a tumor marker, a protein or apeptide capable of specifically recognizing an antibody against theprotein of the present invention or a partial peptide thereof. Morespecifically, the present invention provides a tumor marker comprisingthe protein consisting of amino acid sequence shown in SEQ ID NO: 2 or apartial peptide thereof of the present invention.

Examples of the partial peptide of the protein of the present inventionthat serves as a component of tumor markers include polypeptidesconsisting of at least 8, preferably 15, and more preferably 20contiguous amino acids in the amino acid sequence of the protein of thepresent invention. Tumor can be diagnosed by detecting an antibody in asample (e.g., blood, tissue suspected to contain tumor) using a proteinor a peptide (polypeptide) of the present invention as a diagnosticagent. The method for preparing the protein of the present invention andpeptides are as described in 1) and 2) above.

Specifically, an antibody against PBF can be detected by collectingblood or obtaining a tissue sample suspected to be affected by biopsy orthe like, preparing a protein therefrom in a conventional manner, andconducting detection by a known method such as Western blotting, ELISA,or the like using the protein of the present invention or a peptideabove as a probe.

Diagnostic method of tumor can be done by determining the differencebetween the amount of anti-PBF antibody and that in the correspondingnormal tissue. The difference in the amount of protein includes thecases where the protein is present or absent, and the quantity ofprotein differs by at least 2-fold, preferably 3-fold. Specifically,since the expression of PBF gene is induced in tumors (sarcomas, renalcancer), a test subject is suspected to be affected by tumor, if atissue of the test subject contains antibodies against the expressionproducts (PBF) of said gene and the quantity of said anti-PBF antibodyis determined to be at least 2-fold more preferably 3-fold compared tothat in normal tissue.

10-4) Tumor Marker Related to HLA Tetramer

The present invention also provides an HLA tetramer comprising a peptideof the present invention and an HLA antigen, and a tumor markercomprising the said HLA tetramer.

The term “HLA tetramer” herein used means a tetramer whereinbiotinilated HLA monomers, which monomer is a complex formed byassociation of α-chain of HLA antigen and β2 microglobulin with apeptide (antigen peptide), are allowed to bind to avidin so as to form atetramer (Science 279: 2103-2106 (1998); Science 274: 94-96 (1996)). Atpresent, HLA tetramers comprising a variety of antigen peptides arecommercially available (e.g., Hayashibara Biochemical Laboratories,Inc.), and tetramers comprising a peptide of the present invention andan HLA antigen can easily be prepared.

Examples of specific tetramers include tetramers comprising an antigenpeptide consisting of an amino acid sequence shown in any one of SEQ IDNO: 6-45 and HLA-A24 antigen, and tetramers comprising an antigenpeptide consisting of an amino acid sequence shown in any one of SEQ IDNO: 46-55 and HLA-B55 antigen.

The HLA tetramers are preferably fluorescently labeled so as tofacilitate the selection or detection of bound CTLs by a know detectionmethod such as flow cytometry, fluorescence microscope, or the like.Specifically, tetramers can be labeled with, for example, phycoerythrin(PE), fluoresce in isothiocyanates (FITC), pyridazine chlorophyllprotein (Percy), or the like.

HLA-A24 antigen (α-chain of HLA-A24 antigen) as a component of HLAtetramers can readily be cloned by a conventional method such as PCR onthe basis of known base sequence of HLA-A2402 gene that is disclosed inCancer Res., 55: 4248-4252 (1995) and Genbank Accession No. M64740.Also, HLA-B55 antigen can be cloned on the basis of known base sequenceof HLA-B5502 gene (GenBank Acc. No. M77777, J. Immunol., 148(4),1155-1162 (1992)).

It is preferred that β2 microglobulin, a component of tetramers, ishuman β2 microglobulin. Said human β2 microglobulin can readily becloned by a conventional method such as PCR on the basis of known basesequence of human β2 microglobulin (GenBank Acc. No. AB021288).

Process for preparing HLA tetramers comprising the above components ofHLA tetramers is well known in the art (e.g., Science 279: 2103-2106(1998); Science 274: 94-96 (1996), etc.). The preparation will behereinafter described briefly taking HLA-A24 antigen as an example.

First, both expression vectors each containing HLA-A24 α-chain and β2microglobulin are introduced into E. coli or mammalian cells capable ofexpressing a protein, and the cells are cultured for expression. Forthis step, E. coli (e.g. BL21) is preferred. The resultant monomerHLA-A24 complex and a peptide of the present invention are mixed to forma soluble HLA-peptide complex. The HLA-peptide complex is biotinylatedwith BirA enzyme at the C-terminal sequence of HLA-A24 α-chain. When thebiotinylated HLA-peptide complex is mixed with fluorescently labeledavidin at molar ratio of 4:1, HLA tetramers are formed. It is preferredto conduct gel filtration for purifying protein in each step above.

10-5) Detection Method for Tumor (Diagnostic Method)

The present invention provides a detection method for tumors (diagnosticmethod) utilizing the aforementioned tumor marker of the presentinvention.

Specifically, the detection method (diagnostic method) of the presentinvention comprises collecting blood from a test subject or isolating aspecimen by biopsy or the like from his/her tissue suspected to beaffected; detecting and measuring the expression level of PBF gene,quantity of PBF protein encoded by the gene, quantity of antibodyagainst said PBF protein, or quantity of CTL capable of recognizing acomplex between HLA antigen and tumor antigen peptide from said PBFprotein; and evaluating whether or not the test subject has a tumor suchas sarcoma, renal cancer, or the like, on the basis of the measurements.In addition, according to the detection method (diagnostic method) ofthe present invention, it is possible to detect (diagnose) whether atherapeutic agent administered to a tumor patient for amelioration ofthe tumor can actually bring about improvement of disease or not, or theextent of improvement. Furthermore, the detection method (diagnosticmethod) of the present invention can be used for selecting tumorpatients possibly adaptable to a medicine comprising as an activeingredient a protein, peptide or nucleic acid of the present invention,or the evaluation of the therapeutic effect of said medicine.

The detection method of the present invention comprises the followingsteps (a), (b) and (c):

(a) bringing a sample from living body of the test subject into contactwith a tumor marker of the present invention;

b) measuring expression level of PBF gene, quantity of PBF protein,quantity of anti-PBF antibody, or quantity of CLT capable of recognizinga complex between tumor antigen peptide from PBF and HLA antigen in thesample, and

(c) evaluating whether or not the test subject has a tumor on the basisof measurements in (b).

The sample from a living body herein used includes a sample that can beprepared from tissues such as a tissue suspected to contain tumor,surrounding tissues, or blood, of a living body of test subject.Specifically, such sample includes (i) RNA-containing sample that can beprepared from the above tissue, (ii) polynucleotide-containing samplethat can further be prepared from the sample of (i), (iii) protein- orantibody-containing sample that can be prepared from above tissue, or(iv) peripheral-lymphocyte-containing sample that can be prepared fromthe above tissue.

The diagnostic method of the present invention can be performed asillustrated below depending on the sample from a living body as a targetof measurement.

10-5-1) Method of Measurement Wherein the Target Sample is RNA

When the measurement is directed to RNA, the detection of tumor can beconducted by a process comprising the following steps (a), (b) and (c):

(a) hybridizing RNA prepared from a tissue sample of a test subject orcomplementary polynucleotide transcribed therefrom to a tumor marker ofthe present invention (a polynucleotide comprising at least 15contiguous nucleotides in the base sequence of a polynucleotide of thepresent invention and/or a polynucleotide complementarily thereto);

b) measuring RNA or a complementary polynucleotide transcribed therefromhybridized with the tumor marker using as an index said tumor marker;and

(c) evaluating whether or not the test subject has a tumor on the basisof measurements in (b).

When the RNA is the target sample of measurement, the present detectionmethod (diagnostic method) can be achieved by detecting and measuringthe expression level of PBF gene in the RNA. Specifically, the methodcan be carried out by a known method such as Northern blotting, RT-PCR,DNA chip analysis, in situ hybridization, or the like using anaforementioned tumor marker of the present invention (a polynucleotidecomprising at least 15 contiguous nucleotides in the base sequence of apolynucleotide of the present invention and/or a polynucleotidecomplementary thereto) as a primer or a probe.

In case of Northern blotting, the tumor marker of the present inventionis used as a probe, and the presence/absence or the extent of expressionof PBF gene in RNA can be detected and measured. Specifically, themethod comprises, for example, labeling a tumor marker (complementarystrand) of the present invention with a radioisotope (³²P, ³³P, etc.,RI) or a fluorescent substance, hybridizing the labeled marker to RNAhaving been prepared from a tissue of a test subject and transferredonto a nylon membrane or the like in a conventional manner, anddetecting and measuring the double-strand between the tumor marker (DNA)and RNA on the basis of the signal from the label attached to the tumormarker (RI or fluorescent substance) with a radiation detector(BAS-1800II, Fuji Photo Film., Inc.) or a fluorescence detector.Alternatively, it can be achieved by a method comprising labeling atumor marker (probe DNA) with AlkPhos Direct Labelling and DetectionSystem (Amersham Pharmacia Biotech) in accordance with the protocolattached thereto; hybridizing the labeled marker to RNA originated froma tissue of a test subject, and detecting and measuring a signal fromthe label on the tumor marker using Multi-biomeasure STORM860 (AmershamPharmacia Biotech).

In the case of RT-PCR, tumor marker of the present invention is used asa primer, and the presence/absence or the extent of expression of PBFgene in RNA can be detected and measured expression. Specifically, themethod comprises, for example, preparing cDNA from RNA originated fromtissue of a test subject in a conventional manner, hybridizing a pair ofprimers (positive strand hybridizing to the above-mentioned cDNA andreverse strand capable of binding to said +-strand) having been preparedfrom the tumor marker of the present invention in order to amplify thetarget region corresponding to PBF gene, conducting PCR in aconventional manner, and detecting the amplified double-stranded DNA.The detection of the amplified double-stranded DNA can be achieved by,for example, a method wherein the PCR is conducted with a primerpreviously labeled with RI or a fluorescent substance and the resultantlabeled double-stranded DNA is detected; a method wherein the resultantdouble-stranded DNA is transferred onto a nylon membrane or the like,which is then detected by hybridizing with a labeled tumor marker thatserves as a probe; or the like. The resultant labeled double strandedDNA products may be measured using Agilent 2100 Bioanalyser (YokogawaAnalytical Systems Inc.) or the like. The measurement may also beachieved by preparing RT-PCR reaction solution using SYBR Green RT-PCRReagents (Applied Biosystems) according to the protocol attachedthereto, conducting the reaction using ABI PRISM 7700 Sequence DetectionSystem (Applied Biosystems), and detecting the reaction products.

In the case of DNA chip analysis, an example of method comprisespreparing a DNA chip on which the tumor marker of the present inventionused as a DNA probe (single- or double-strand) is attached, hybridizingthereto cRNA having been prepared from RNA originated from tissue of atest subject in a conventional manner, detecting a double stand formedbetween DNA and cRNA after binding to a labeled probe prepared from thetumor marker of the present invention.

10-5-2) Method of measurement wherein the target sample is protein

When the target sample of measurement is protein, the detection method(diagnostic method) of tumor of the present invention can be conductedby a process comprising the following steps (a), (b) and (c):

(a) allowing proteins prepared from a tissue sample of a test subject tobind to a tumor marker of the present invention related to antibody(antibody recognizing PBF);

b) measuring proteins bound to the tumor marker using as an index saidtumor marker; and

(c) evaluating whether or not the test subject has a tumor on the basisof measurements in (b).

Specifically, the detection method includes a method wherein PBF isdetected and measured by a known method such as Western blotting usingantibody (antibody recognizing PBF) as a tumor marker of the presentinvention.

Western blotting can be carried out using, as the primary antibody, atumor marker, and then, as the second antibody, a labeled antibody(antibody capable of binding to the primary antibody) labeled with aradio isotope (e.g., - 125I, RI), fluorescent substance, enzyme (e.g.,horse radish peroxidase, HRP), or the like, and detecting and measuringthe signal from RI or fluorescent substance of resultant labeledcompounds using radiation detector (BAS-1800II, Fuji Photo Film., Inc.,etc), fluorescence detector, or the like. It can also be achieved by amethod wherein, after using the tumor marker of the present invention asa primary antibody, the detection is carried out with ECL Plus WesternBlotting Detection System (Amersham Pharmacia Biotech & Science)according to the protocol attached thereto, and measurement withMulti-biomeasure STORM860 (Amersham Pharmacia Biotech).

10-5-3) Method of Measurement Wherein the Target Sample is Antibody

When the target sample of measurement is antibody contained in proteins,the detection method (diagnostic method) of tumor of the presentinvention can be performed by detecting the anti-PBF antibody in atissue sample of living body, and measuring the quantity thereof.Specifically, it can be carried out in a similar manner to thatdescribed in 10-5-2) above using a tumor marker of the present inventionrelated to protein or peptide.

10-5-4) Method of Measurement Wherein the Target Sample is CTL Specificfor Tumor Antigen

When the target sample of measurement is tumor-antigen-specific CTLs inperipheral blood lymphocytes, the detection method (diagnostic method)of tumor of the present invention can be performed by detectingPBF-specific CTLs in a sample of living body, and measuring the quantitythereof. Specifically, it can be carried out by preparing a tetramer(HLA tetramer) by complexing an HLA antigen fluorescently labeledaccording to a method described in a literature (Science, 274: p 94,1996) with a peptide of the present invention, and subjecting thetetramer to the detection of CTLs specific for antigen peptides inperipheral blood lymphocytes of a patient suspected to have a tumorusing flow cytometry.

10-5-5) Diagnosis of Tumors

Diagnosis of tumor can accomplished by measuring the expression level ofPBF gene, quantity of PBF protein that is an expression product of thegene, quantity of anti-PBF antibody, or quantity of PBF-specific CTLs inblood or a tissue suspected to have a tumor of a test subject.Optionally, diagnosis may be carried out by comparing the expressionlevel of said gene or quantity of said protein with that in thecorresponding normal tissue.

The comparison of quantity (level) of a gene, protein, antibody or CTLbetween a tissue of a test subject and the corresponding normal tissuecan be carried out by conducting the measurement on the sample from thetest subject and that from a normal subject in parallel. When theparallel measurement is not performed, a mean value or statisticalintermediate value of the results regarding expression level of PBFgene, quantity of PBF, quantity of anti-PBF antibody or quantity ofPBF-specific CTL of a normal subject is used as the value of normalsubject, which results have been obtained by conducting measurementunder uniform conditions for plural (at least 2, preferably 3, morepreferably 5) normal tissues.

Diagnosis whether or not the test subject has a tumor can be carried outon the basis of criterion that the expression level of PBF gene,quantity of PBF, quantity of anti-PBF antibody or quantity ofPBF-specific CTL in a tissue of a test subject is at least 2-fold,preferably 3-fold higher compared to that in the corresponding tissue ofa normal subject.

EXAMPLES

The present invention is further illustrated by the following examples,but is not limited by these examples in any respect.

Example 1 Establishment of Osteosarcoma Cell Line and Cytotoxic T

Lymphocytes (CTLs) Cell Line Thereto

A osteosarcoma cell line was established from biopsy osteosarcoma tissueof osteosarcoma patient and named as OS2000. Peripheral-bloodmononuclear cells were separated from blood obtained from the samepatient by density centrifugation using Lymphoprep (Nycomed), andcultured while stimulating the cells 6 times per every 10 days by addingOS2000 previously inactivated by radiation. CD8-positive cells were thenenriched with magnetic beads to which anti-CD8 antibodies are bound(Macs, Miltenyi) and CTL cell lines were established by limitingdilution method (Int. J. Cancer, 39, 390-396, 1987, N. Eng. J. Med, 333,1038-1044, 1995). Screening was conducted using as an index thecytotoxic activity on OS2000 to obtain three CTLs. Among them, a CTLnamed TcOS2000cl-303 was selected and used in the following experiments.It was confirmed that HLA class I molecules for OS2000 include antigensof HLA-A*2402, -B*5502, -B40 and Cw1 types.

Example 2 Screening of Gene Encoding Novel Tumor Antigen Protein

1) Screening Using Cells Transformed with HLA-B55 Gene

From 052000 was prepared mRNA with First track (Invitrogen). A cDNAlibrary was then constructed by preparing a cDNA from the mRNA usingSuperscript Choice System for cDNA Synthesis (Gibco-BRL) andincorporating into an expression vector pCEP4 (Invitrogen).

On the other hand, HLA-B* 5502 gene (GenBank Acc. No. M77777; J.Immunol., 148(4), 1155-1162 (1992)) isolated from OS2000 cells by PCRwas introduced into 293-EBNA cells (Invitrogen) to yield 293-EBNA-B55.

The first screening involved the following procedures. A pool of about100 cDNA clones (100-200 μg) was introduced into 293-EBNA-B55 cellshaving been plated into a 96-well microplate at 8×10⁴ cells/well bylipofection method (Lipofectamine 2000, Invitrogen). Pools of respectivecDNA clones used for introduction were stored separately.Twenty-four-hour later, TcOS2000cl-303 cells (8×10⁴) were added andcultivation continued for another 24 hours, when the culturedsupernatant was recovered. The reactivity of CTL was evaluated bydetermining LDH released in the supernatant by cytotoxic effects usingLDH Cytotoxicity Detection Kit (TAKARA BIO). Among 1000 wells, an wellnamed “IB9” containing the most potent CTL reactivity was selected.Eighty pools of cDNA clones (8-12 cDNA clones/pool) were prepared from400 cDNAs prepared from the cDNA clone pool that has been used for IB9gene introduction, and introduced into 293-EBNA-B55 cells in the samemanner as the above, and the second screening was conducted using as anindex the reactivity of TcOS2000cl-303 cells. As a result, thereactivity was detected in 20 wells. A cDNA clone from the cDNA clonepool in each well of 20 wells was introduced into 293-EBNA-B55 cells ina similar manner to the above and the third screening was conductedusing as an index the reactivity of TcOS2000cl-303 cells. As a result,the reactivity was detected in 4 wells. The base sequence analysis ofeach cDNA in 4 wells revealed that these wells contained the same cDNA,which clone was named as IB9.1H4.

2) Screening Using Cells Transfected with HLA-A24 Gene

In a similar manner to the HLA-B* 5502 gene above, an HLA-A* 2402 gene(Cancer Res., 55:4248-4252 (1995), GenBank Accession No. M64740) wasintroduced into 293-EBNA cells (Invitrogen) to prepare 293-EBNA-A24.Screening was carried out a similar manner to the above where293-EBNA-B55 cells were used. Thus, the well 2E4 was obtained by thefirst screening; 22 reactive wells were obtained from the 2E4 by thesecond screening; and 4 wells were selected by the third screening. Thebase sequence analysis of cDNA of 4 wells reveled that they contain thesame cDNA as cDNA clone 1B9.1H4 that was selected by HLA-B* 5502.

3) Analysis of cDNA Clone 1B9.1H4

1B9.1H4 cDNA was introduced into 293-EBNA-B55 or 293-EBNA-A24 andreactivity of TcOS2000cl-303 to the transfected cell expressing thetransgene was measured by LDH release assay (determination of releasedLDH). The results are shown in FIG. 1. The reactivity of TcOS2000cl-303was also measured by ⁵¹Cr release assay (J. Immunol., 159: 4753, 1997),and the results are shown in FIG. 2 (A, B). TcOS2000cl-303 recognizedthe cells expressing the introduced cDNA clone 1B9.1H4 and exertedspecific cytotoxicity. These results show that cDNA clone 1B9.1H4encodes a gene of tumor antigen protein that is recognized byTcOS2000cl-303.

Base sequence of cDNA 1B9.1H4 was determined using BigDye TerminatorCycle Sequencing Ready Reaction Kit (Applied Biosystems). The basesequence is shown in SEQ ID NO:3. The full length cDNA was 1901 basepairs in length. The base sequence of SEQ ID NO: 3 was compared withknown sequences using publicly available databases and proved to be, forthe most part, identical with that of a gene encoding papillomavirusbinding factor (PBF), which is also called as papillomavirus regulatoryfactor (PRF-1) registered as GenBank Accession No. AF263928 (Virology293, 103-117 (2002)). The base sequence encoding PBF and amino acidsequence corresponding thereto are shown in SEQ ID NO: 1 and 2,respectively.

It was then examined whether or not PBF itself has an activity(reactivity to CTL) as a tumor antigen protein similar to that ofaforementioned 1B9.1H4. PBF cDNA was amplified by PCR using cDNAprepared from RNA that has been extracted from OS2000 cells and primersshown in SEQ ID NO: 4 and SEQ ID NO: 5. A PBF gene expression vectorconstructed by inserting the amplified fragment into expression vectorpCEP4 was introduced into the aforementioned 293-EBNA-B55 or293-EBNA-A24, and reactivity of TcOS2000cl-303 to the transfected cellsexpressing the transgene was measured by LDH release assay. The resultsare shown in FIGS. 3 and 4. As is clear from the figures, TcOS2000cl-303also showed cytotoxic effect on the cells having been transformed with aPBF gene expression vector and expressing the gene. This demonstratedthat PBF is a tumor antigen protein that is recognized byTcOS2000cl-303.

Example 3 Identification of Antigen Peptide Binding to HLA-B*5502

Antigen peptide regions that bind to HLA-B* 5502 was identified fromvarious peptides synthesized on the basis of the amino acid sequence(SEQ ID NO: 2) of tumor antigen protein PBF using as an index thereactivity of TcOS2000cl-303. 293-EBNA-B55A cells (8×10⁴) were pulsedfor 1 hour with a peptide having an amino acid sequence (Cys Thr Ala CysArg Trp Lys Lys Ala Cys Gln Arg, SEQ ID NO: 46) corresponding to theamino acid sequence at position 499-510 of SEQ ID NO: 2, co-culturedwith TcOS2000cl-303 cells (4×10⁴) and the reactivity was assessed by LDHrelease assay in the same manner as that described in Example 2. Theresults are shown in FIG. 5. As can be seen, TcOS2000cl-303 cells werenot reactive to pepitde-unpulsed 293-EBNA-B55 cells but reacted topeptide-pulsed 293-EBNA-B55 cells, wherein the potpie corresponds to SEQID NO: 46. Accordingly, it became clear that the peptide of SEQ ID NO:46 contains a region capable of binding to HLA-B* 5502.

Example 4 Expression Analysis of PBF Gene in Various Cells and Tissues

The expression of a gene encoding tumor antigen protein PBF in varioustypes of cell and tissue was examined by RT-PCR. cDNA was prepared fromRNA extracted from respective cells with Isogen regent (Nippon Gene)using oligo dT primer. PCR was carried out using primers shown in SEQ IDNO: 4 and SEQ ID NO: 5 to amplify PBF cDNA. The PCR products were thenseparated electrophoretically and analyzed. As a positive control,expression of GPDH gene was examined by RT-PCR method in the same mannerabove. The results are shown in FIG. 6. Expression of PBF gene was notdetected in normal peripheral blood lymphocytes or 293-EBNA cells usedfor screening, but was confirmed in many cells including OS2000originated from sarcomas.

Example 5 Expression Analysis of PBF Gene in Renal Cancer Tissue

Expression of a gene encoding tumor antigen protein PBF in renal cancertissue and normal renal tissue was analyzed using DNA chips. The DNAchip analysis was carried out by a usual method described in WO03/048359 and the like. As a result, the median expression amount of 91renal cancer tissues was 780 while that of 67 normal renal tissues was89, indicating that the expression of PBF gene is increased in renalcancer tissues by about 8.7-fold in a cancer specific manner.

Example 6 Identification of Anti-Tumor Peptides Capable of Binding toHLA-A* 2402

A peptide consisting of amino acid sequence shown in SEQ ID NO: 6, 7, 8,10, 26, 27, 28, 29, 30 or 32 which comprises an HLA-A24 binding motif inthe amino acid sequence (SEQ ID NO: 2) of tumor antigen protein PBF wassynthesized. Identification of tumor-antigen peptides capable of bindingto HLA-A*2402 was carried out using as an index the reactivity ofTcOS2000cl-303. That is, a tumor-antigen peptide capable of binding toHLA-A*2402 can be identified by co-culturing TcOS2000cl-303 cells with293-EBNA-A24 cells pulsed with any one of peptides above, and examiningthe reactivity of CTLs by LDH release assay in a similar manner toExample 2.

INDUSTRIAL APPLICABILITY

The present invention provides use of tumor-antigen protein PBF and agene encoding the same as an inducer of CTL, or the like. The inducer ofCTL of the present invention is useful in treatment of patientssuffering from sarcomas or renal cancer.

1. An inducer of cytotoxic T cell (CTL) comprising as an activeingredient a protein which comprises the same or substantially the sameamino acid sequence as that shown in SEQ ID NO:
 2. 2. An inducer of CTLcomprising a polynucleotide encoding a protein comprising the same orsubstantially the same amino acid sequence as that shown in SEQ ID NO:2.
 3. The inducer of CTL of claim 2, wherein the polynucleotide is apolynucleotide comprising a base sequence shown in SEQ ID NO: 1,position 337-1878 of SEQ ID NO: 1 or SEQ ID NO:
 3. 4. A method forproducing an antigen-presenting cell comprising the step of bringing acell having antigen-presenting ability into contact with (a) or (b) invitro: (a) a protein comprising the same or substantially the same aminoacid sequence as that shown in SEQ ID NO: 2; (b) a nucleic acidcomprising a polynucleotide encoding the protein of (a).
 5. A method forinducing a CTL comprising the step of bringing peripheral lymphocytecells into contact with (a) or (b) in vitro: (a) a protein comprisingthe same or substantially the same amino acid sequence as that shown inSEQ ID NO: 2; (b) a nucleic acid comprising a polynucleotide encodingthe protein of (a).